Methods and compositions for nucleic acid amplification

ABSTRACT

In some embodiments, the disclosure relates generally to methods, as well as related compositions and kits for recombinase-mediated nucleic acid amplification, such as recombinase-polymerase amplification (RPA), of a nucleic acid template using at least one blocked primer that contains a 5′ domain, at least one nucleotide that is cleavable by an RNase H enzyme, a 3′ domain, wherein the primer is not extendable by a polymerase, and wherein the 3′ domain has a length of 7-100 nucleotides, for example 10-30 nucleotides. These methods and the use of a blocked primer reduce or eliminate non-specific amplification products, such as primer dimers, which are generated in RPA reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/308,803, filed Dec. 10 2018, which is a U.S. National Applicationfiled under 35 U.S.C. § 371 of International Application No.PCT/US2017/036842, filed on Jun. 9, 2017, which claims the benefit ofpriority under 35 U.S.C. § 119 to U.S. provisional application No.62/348,402, filed Jun. 10, 2016; the disclosures of all theaforementioned applications are incorporated by reference in theirentireties.

Throughout this application, various publications, patents, and/orpatent applications are referenced. The disclosures of the publications,patents and/or patent applications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“LT01140US_ST25.txt” created on Dec. 10, 2018 and is 6,000 bytes insize. The sequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

BACKGROUND

Nucleic acid amplification is very useful in molecular biology and haswide applicability in practically every aspect of biology, therapeutics,diagnostics, forensics and research. Generally, amplicons are generatedfrom a starting template using one or more primers, where the ampliconsare homologous or complementary to the template from which they weregenerated. Multiplexed amplification can also streamline processes andreduce overheads. This application relates to methods and reagents fornucleic acid amplification and/or analysis using cleavable primers.

One example of such amplification is Recombinase PolymeraseAmplification (RPA) which is a DNA amplification process that utilizesenzymes that hybridize oligonucleotide primers to their complementarypartners in DNA (e.g., duplex DNA) followed by isothermal amplification.RPA offers a number of advantages over traditional methods of DNAamplification. These advantages include the lack of a need for anyinitial thermal or chemical denaturation, the ability to operate at lowconstant temperatures (e.g., isothermal conditions) without a need forabsolute temperature control, as well as the observation that completereactions (lacking target) can be stored in a dried condition. Thesecharacteristics demonstrate that RPA is a uniquely powerful tool fordeveloping portable, accurate, and instrument-free nucleic aciddetection tests. However, use of standard primers in RPA methods mayresult in nonspecific amplification product and/or primer dimerproducts, which reduce the efficiency of the reaction especially in theinstance of next gen sequencing. Furthermore, primer design constraintsare a drawback of RPA.

SUMMARY

Herein are provided blocked primers (e.g., oligonucleotide primers)containing a ribose base separating a 5′ domain and a 3′ domain of theprimers, optionally for use in amplification reactions, especiallyisothermal reactions such as recombinase polymerase amplification (RPA).The ribose base moiety can be cleaved by certain endonuclease enzymessuch as RNase H. The use of at least one such blocked primer (e.g.,forward and/or reverse oligonucleotide primers), and an endonucleasethat cleaves ribobase(s) (e.g. RNase H) after primer binding to atemplate DNA, reduces nonspecific amplification products providing animproved method for amplification of nucleic acid. Specific andsurprising configurations for such primers have been identified, thatprovide effective primers for reactions that involve endonucleasecleavage of the primers, such as amplification reactions. Amplificationreactions can include but are not limited to PCR (Polymerase ChainReaction), HCR (Hybridization Chain Reaction), RCA (Rolling CircleAmplification), RPA (Recombinase Polymerase Amplification), LAMP (Loopmediated isothermal amplification), HDA (Helicase DependentAmplification), cluster-generation methods such as bridge amplification(e.g. U.S. Pat. Appln. No. 2008/0009420 (Schroth et al.)) and“template-walking” (e.g. U.S. Pat. Appln. No. 2012/083189 (Li et al.)).

Methods, reagents and products of nucleic acid amplification and/oranalysis are provided herein. In some embodiments, the present teachingsprovide compositions, systems, methods, apparatuses and kits for nucleicacid amplification.

Methods are provided for cleaving a double-stranded nucleic acid with anendonuclease, comprising the steps of (1) forming a reaction mixturecomprising template nucleic acids (e.g., nucleic acid templates) andprimers which include a cleavable moiety (e.g., a ribose base), whereinthe primers are at least partly complementary to the template nucleicacid, (2) exposing the resulting mixture to conditions suitable forhybridization between the primers and the template nucleic acids, and(3) cleaving the primers at the cleavable moiety with the endonuclease,where the primers are at least partly hybridized to the template nucleicacids. In some embodiments, the endonuclease selectively cleaves theprimers. In some embodiments, the endonuclease does not cleave theprimers at other nucleotide positions. In some embodiments, theendonuclease cleaves a significant fraction of primers. In someembodiments, the endonuclease cleaves the primers less efficiently atelevated temperatures. In some embodiments, the endonuclease is RNaseH2. In some embodiments, the cleavable moiety is a ribose base (e.g., aribonucleotide). In some embodiments, the cleaving step is performed ata temperature below 60° C. (e.g. at room temperature or about 20-50° C.,20-30° C., 20-40° C., 25-40° C., 30-40° C., 35-40° C., or 40-50° C.). Insome embodiments, the reaction mixture is contacted with amplificationreagents and/or subjected to amplification conditions. In someembodiments, the reaction mixture further comprises a plurality ofsecond primers that are reverse-complementary to the template nucleicacid, and the second primers optionally comprise a cleavable moiety(e.g., a ribose base), where the cleavable moiety is situated more than5 nucleotides away from the 3′ end of the oligonucleotide (e.g., atleast 7, 10, 12, 15 or 20 nucleotides).

In some embodiments, the disclosure relates to methods for cleaving oneor more blocked primers, comprising the steps of forming a reactionmixture by combining a nucleic acid template, a forward primer, areverse primer, an RNase H enzyme, and optionally a source of reactivenucleotides such as dNTPs, and optionally a buffer, wherein the forwardprimer binds to a forward primer binding site on the nucleic acidtemplate and the reverse primer binds to a reverse primer binding siteon the nucleic acid template, wherein one or both of the forward orreverse primers is blocked, and wherein the blocked primer comprises a5′ domain and a 3′ domain separated by at least one cleavable nucleotide(e.g., a ribobase), wherein the 5′ domain is at least 10 nucleotides inlength (e.g. 10 to 100 nucleotides in length) and the 3′ domain is atleast 10 nucleotides in length (e.g. 10 to 100, 10 to 90, 10 to 80, 10to 75, 10 to 60, or 10 to 50 nucleotides in length); and optionallyincubating the reaction mixture under substantially isothermal, orisothermal, amplification conditions between 20° C. and 50° C. (e.g. 25°C. and 40° C., 30° C. and 40° C., or 35° C. and 40° C.) for 10 minutesto 120 minutes, thereby amplifying the nucleic acid template. In anembodiment the reaction mixture optionally comprises at least one ormore of: a polymerase, a recombinase, a single-stranded binding protein,or a recombinase loading protein.

In some embodiments, the disclosure relates to methods for cleaving ablocked primer, comprising the steps of forming a reaction mixture bycombining a nucleic acid template having a forward primer bindingsequence and a reverse primer binding sequence, and a blocked forwardprimer (i.e. non-extendable primer; i.e. forward primer that is notextendable by a polymerase), a reverse primer which is optionallyblocked, an RNase H enzyme, and optionally a buffer comprising adivalent cation, wherein the forward primer binding sequence iscomplementary or identical to at least a portion of the blocked forwardprimer and the reverse primer binding sequence is complementary oridentical to at least a portion of the blocked reverse primer, andwherein the blocked forward primer and the blocked reverse primercomprise a 5′ domain and a 3′ domain separated by at least one cleavablenucleotide (e.g. comprising a ribobase), wherein the 5′ domain is atleast 10 (e.g. 10 to 70, 10 to 60, 10 to 50, or 10 to 40) nucleotides inlength and the 3′ domain is at least 10 (e.g. 10 to 70, 10 to 60, 10 to50, 10 to 40 or 10 to 25) nucleotides in length; and optionallyincubating the reaction mixture under substantially isothermalamplification conditions (e.g., between 20° C. and 50° C. (e.g. 25° C.and 40° C., 30° C. and 40° C., or 35° C. and 40° C.)) for 15 minutes to60 minutes, thereby amplifying the nucleic acid template. In someembodiments, the reaction mixture optionally comprises at least one ormore of: a polymerase, a recombinase, a single-stranded binding protein,or a recombinase loading protein.

In some embodiments, the disclosure relates to methods for nucleic acidamplification, comprising the steps of forming a reaction mixture bycombining at least two different polynucleotide templates comprisingboth a first primer binding sequence and a second primer bindingsequence, a recombinase, a recombinase accessory protein, a polymerase,a first blocked universal primer, a second optionally blocked universalprimer, an RNase H enzyme, and optionally dNTPs and a buffer, whereinthe reaction mixture is in contact with a support having the firstblocked universal primer bound (e.g., immobilized) thereto, wherein thefirst primer binding sequence is complementary or identical to at leasta portion of the first blocked universal primer and the second primerbinding sequence is complementary or identical to at least a portion ofthe second blocked universal primer, and wherein the first blockeduniversal primer and the second blocked universal primer comprise a 5′domain and a 3′ domain separated by a nucleotide comprising a ribobase,w wherein the 5′ domain is 10 to 70, 10 to 60, 10 to 50, or 10 to 40nucleotides in length and the 3′ domain is 10 to 70, 10 to 60, 10 to 50,10 to 40 or 10 to 25 nucleotides in length; and forming at least twosubstantially monoclonal nucleic acid populations by using thepolymerase to amplify each of said at least two different polynucleotidetemplates onto different sites on the solid support, within the samereaction mixture of step (a) under substantially isothermal conditions.The amplified monoclonal nucleic acid populations may be sequenced.

In some embodiments, the blocked forward primer and the blocked reverseprimer comprise a 5′ domain and a 3′ domain separated by a nucleotidecomprising a ribobase, wherein the 5′ domain is 10 to 100 nucleotides inlength and the 3′ domain is 11 to 30 nucleotides in length. Inembodiments the 5′ domain is 15 to 25 nucleotides in length or greaterthan 25 nucleotides. In some embodiments, the 5′ domain 15 to 50nucleotides in length. In some embodiments, the ribobase is rU, rG, rCor rA.

In some embodiments, the 3′ domain is 14 to 25 nucleotides in length or15 to 25 nucleotides in length. In some embodiments, a 3′ nucleotide ofthe 3′ domain of the forward primer is mismatched to the forward primerbinding sequence.

In some embodiments the recombinase is selected from the groupconsisting of uvsX, RecA, RadA, RadB, Rad 51, a homologue thereof, afunctional analog thereof and a combination thereof. In someembodiments, the reaction mixture comprises uvsY accessory protein anduvsX recombinase.

In some embodiments, the RNase H enzyme is RNase HII. In someembodiments, the RNase H enzyme is RNase HII and the incubatingtemperature is between 35° C. and 42° C.

In some embodiments, the RNase H enzyme is present at a concentrationfrom 5 U to 200 U/50 μL, or from 10 to 90 U/50 μL.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a non-limiting schematic of RPA with RNase H enzyme cleavagemethods. A standard RPA is shown in the top portion of FIG. 1. An RPAwith RNase H cleavage is shown in the lower portion of FIG. 1. The twoinverted ovals around single chain represent recombinase bound tooligonucleotide. The open rectangles represent single stranded bindingprotein. The solid diamond represents a blocking moiety. The singlevertical oval across two strands represents RNase bound to doublestranded nucleic acid. The ¾ open circle represents a polymerase.

FIG. 2 is a non-limiting schematic of blocked primers and exemplaryprimer configurations, including exemplary 5′ domain and 3′ domainlengths.

FIG. 3 is a photo of a gel showing the results of blocked primerconfiguration screening with V1, V2 and V3 primers by comparing DNAtemplate amplification using the blocked primers.

FIG. 4 is a photo of a gel showing the results of blocked primerconfiguration screening with V4 and V5 primers by comparing DNA templateamplification using the blocked primers.

FIG. 5 is a photo of a gel showing the results of blocked primerconfiguration screening with V4 and V5 primers by comparing DNA templateamplification using the blocked prime with longer exposure to detectionreagent.

FIG. 6 is a photo of a gel showing the results of DNA templateamplification wherein nonspecific amplification using a non-templatecontrol reduced or eliminated using blocked primers of the invention.

FIG. 7 is a photo of a gel showing the results of DNA templateamplification wherein nonspecific amplification is reduced or eliminatedusing blocked primers of the invention.

FIG. 8A is a photo of two gels showing the results of RNase H enzymeunit titration by analyzing DNA amplification using variousconcentrations of RNase H enzyme in an RPA reaction with blockedprimers.

FIG. 8B is a photo of a gel showing the results of RNase H enzyme unittitration by analyzing DNA amplification using various concentrations ofRNase H enzyme in an RPA reaction with blocked primers.

FIG. 8C is a photo of a gel showing the results of DNA amplificationwith a range of amplification reaction temperatures.

FIG. 9 is a photo of a gel showing the results of DNA amplificationusing two different pellet formulations of enzymes rehydrated in an RPAreaction with blocked primers of the invention and regular primercontrols.

FIG. 10 is a list of exemplary blocked primer and adapter sequences.

FIG. 11 is a list of exemplary primer sequences for amplification on asolid surface.

FIG. 12A is a table showing the reaction volumes and times for anamplification sequencing reaction with results shown in FIG. 12B.

FIG. 12B is a table that lists a comparison of DNA templateamplification on a solid support using RNase H cleavable blocked primersof the invention followed by sequencing of the amplified template.

FIG. 12C is a read length histogram for sequencing results using thereaction volumes and times of FIG. 12A.

FIG. 13 is a photo of a gel showing the results of DNA amplificationusing Endo IV endonuclease and an abasic cleavable blocked primer in anRPA reaction.

FIG. 14 is a photo of a gel showing the results of DNA amplificationusing APE 1 endonuclease and an abasic cleavable blocked primer in a RPAreaction as compared to a ribobase cleavable blocked primer and RNaseHII enzyme.

FIG. 15 is a photo of a gel showing the results of DNA amplificationusing the endonuclease APE 1 or Endo IV and an abasic blocked primer ina RPA reaction as compared to a ribobase cleavable blocked primer andRNase HII enzyme and controls.

DETAILED DESCRIPTION

In some embodiments, reducing nonspecific amplification includingreducing primer dimer formation in nucleic acid amplification reactions.In some embodiments, the reduced non-specific amplification can beachieved in an isothermal amplification reaction, for example using arecombinase-mediated amplification reaction such as RPA(recombinase-polymerase amplification). In some embodiments, simplifiedprimer designs can be used for such reactions. In some embodiments, themethods, compositions, and kits use blocked primers that comprise one ormore cleavable moieties (e.g., ribose bases) that separate a 5′ domainand 3′ domain of the primer, wherein an enzyme (e.g., ribo-endonuclease,such as RNase H), cleaves the primer at the cleavable moiety locationthereby removing the blocking moiety. The 5′ domain of the primerremains hybridized to the template nucleic acid while the 3′ domain isremoved. In some embodiments, the methods, compositions and kitsidentify surprising ranges for 5′ and especially 3′ domain nucleotidelengths of the blocked primers. These domain lengths, discussed below indetail, surprisingly result in efficient amplification of the templatenucleic acid and surprisingly reduce or even eliminate primer dimerproduct formation and nonspecific amplification. In some embodiments,the methods include clonal amplification that utilizerecombinase-mediated amplification and the improved blocked primers. Insome embodiments, the methods include using especially effectiveconcentration ranges (excess concentration) for RNase H in suchrecombinase amplification reactions using blocked primers that include aribobase.

In some embodiments, therefor amplifying a nucleic acid template, thatincludes forming a reaction mixture by combining the nucleic acidtemplate (e.g., a template polynucleotide), a polymerase, a recombinase,a forward primer, a reverse primer, wherein at least one of the forwardprimer or the reverse primer is a blocked primer, dNTPs, an RNase Henzyme, and a buffer. In some embodiments, the blocked primer is ablocked forward primer that binds to a forward primer binding site onthe nucleic acid template and the reverse primer binds to a reverseprimer binding site on the reverse complement of the nucleic acidtemplate. The blocked forward primer comprises a 5′ domain and a 3′domain separated by at least one nucleotide comprising a ribobase and ablocking group on the 3′ end of the primer. The reaction mixture, insome embodiments, is incubated under substantially isothermalamplification conditions to amplify the nucleic acid template.

In some embodiments, methods for amplifying nucleic acid template(s)upstream of sequencing methods. Nucleic acid templates for theseembodiments can be at least some, and typically all members of a nucleicacid sequencing template library. In some embodiments, the methodincludes forming a reaction mixture by combining at least two differentpolynucleotide templates comprising both a first primer binding sequenceand a second primer binding sequence, a recombinase, a recombinaseaccessory protein, a polymerase, a first blocked universal primerattached to a support, a second optionally blocked universal primer,dNTPs, an RNase H enzyme, and a buffer, wherein the reaction mixture isin contact with the support, wherein the first primer binding sequenceis complementary or identical to at least a portion of the first blockeduniversal primer and the second primer binding sequence is complementaryor identical to at least a portion of the second blocked universalprimer. The polymerase, by amplifying the at least two differentpolynucleotide templates, forms at least two substantially monoclonalnucleic acid populations onto different sites on the solid support,within the same reaction mixture of the first step under substantiallyisothermal conditions. This multi-clonal population of amplified nucleicacid template may then be used in sequencing workflow methods, such ashigh throughput sequencing methods.

In some embodiments, the methods use recombinase to denature, orpartially denature, double stranded nucleic acid templates, which can becarried out at isothermal conditions with a polymerase, and is referredto as recombinase-polymerase amplification (RPA) (see, e.g.,WO2003072805, hereby incorporated by reference in its entirety). In someembodiments, the partial denaturation and/or amplification, includingany one or more steps or methods described in the teachings herein, canbe achieved using a recombinase and/or single-stranded binding protein.Suitable recombinases include RecA and its prokaryotic or eukaryotichomologues, or functional fragments or variants thereof, optionally incombination with one or more single-strand binding proteins (SSBs). Insome embodiments, the recombinase optionally binds single-stranded DNA(ssDNA) such as the blocked primers, to form a nucleoprotein filamentstrand which invades a double-stranded region of homology on a template.See FIG. 1. This optionally creates a short hybrid and a displacedstrand bubble known as a D-loop. In FIG. 1, the free 3′-end of thehybridized primer after cleavage by the RNase H enzyme is extended byDNA polymerases to synthesize a new complementary strand. Thecomplementary strand displaces the originally-paired partner strand ofthe template as it elongates. In some embodiments, the one or more of apair of blocked primers are contacted with one or more recombinasesbefore being contacted with a template which is optionallydouble-stranded.

In any of the methods described herein, amplification of a templateoptionally comprises contacting at least one blocked primer with atemplate strand, which optionally has a region of complementarity to atleast one blocked primer. After hybridization of the blocked primer tothe template DNA, the blocked primer is cleaved at the cleavable moiety(e.g., ribose base) location thereby liberating the 3′ domain of theblocked primer. The newly formed 3′ end of the 5′ domain of the primeris then extended along the template with one or more polymerases (e.g.,in the presence of dNTPs) to generate a double stranded nucleic acid anda displaced template strand. The amplification reaction can compriserepeated cycles of such contacting and extending until a desired degreeof amplification is achieved, including, In some embodiments,substantially monoclonal amplification of the template DNA. Optionallythe displaced strand of nucleic acid is amplified by a concurrentamplification reaction. Optionally, the displaced strand of nucleic acidis amplified by contacting it in turn with one or more complementaryblocked primers and extending the complementary primer (after cleavagewith an RNase H enzyme) by any strategy described herein. Optionallybefore a blocked primer is contacted with a template nucleic acid, it isfirst contacted with an amplification enzyme (e.g. a recombinase or apolymerase) which binds to the primer. Any amplification enzyme that hasnot associated with the one or more blocked primers is optionallyremoved.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits which utilize at least one blockedRNase cleavable primer in the amplification of template nucleic acid.The blocked primer can be the forward primer, reverse primer or both. Aforward primer and reverse primer typically form a primer pair foramplification. The forward primer binds a forward primer bindingsequence in a forward direction on a forward strand. The reverse primerbinds to a reverse primer binding sequence in a reverse direction on thecomplement strand of the forward strand. The blocked primers can beuniversal primers, which can, for example, bind to a target sequence ina gene or other sequence of interest, bind to a sequence found in aplasmid cloning vector, or in some embodiments, bind to universaladaptors found on or near the ends of template nucleic acids of anucleic acid library. In some embodiments, one or more of the universalprimers does not contain any target (template) specific sequences. Insome embodiments, both the forward and reverse blocked primers of theinvention are universal primers, which hybridize to a universal adaptersequence in the nucleic acid template. See Example 5. In someembodiments, only one of the forward and reverse blocked primers of theinvention is a universal primer. Provided at least one primer is ablocked primer, then the other primers, forward or reverse, can bestandard (non-blocked) primers. In some embodiments, the disclosurerelates generally to methods, as well as related compositions and kits,wherein blocked primers that are cleavable by RNase H. In someembodiments, the blocked primers contain at least four components; a 5′domain, at least one ribobase, which is part of a cleavable segment, a3′ domain and a blocking moiety (See FIG. 2). Such primers can bereferred to herein, for example, as “blocked primers”, “non-extendableprimer” or “blocked RNase cleavable primers.” The ribobase, for examplea ribonucleotide, when the primer is hybridized to a DNA template, issusceptible to cleavage by ribo-endonucleases, thereby separating the 5′domain and the 3′ domain. See FIG. 2. Surprisingly, primers thatincluded long domains, especially long 3′ domains, were effective inreducing or eliminating primer dimer product formation. In someembodiments, the 3′ domain is at least 7, 10, 12 or 14 nucleotides long,e.g. 14 to 30 nucleotides in length. In some embodiments, the blockedprimer is between 15 and 200 nucleotides long, and includes a ribobasethat is more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotidesaway from the 3′ terminus of the blocked primer, referred to in thealternative embodiment herein, as the oligonucleotide. It is noteworthythat the Alternative Embodiments herein include oligonucleotides thatare not extendable by a polymerase (i.e. blocked oligonucleotides). Suchblocked oligonucleotides include blocked primers that are cleavable byRNase H, as well as other oligonucleotides that are cleavable by otherenzymes, as provided in the Alternative Embodiments section herein.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits, which use at leastoligonucleotide that is not extendable by a polymerase, such as oneblocked primer that is cleavable by RNase H. In some embodiments, theblocked primer is a blocked forward primer. In some embodiments, theblocked primer is a blocked reverse primer. In some embodiments theblocked primers are complementary or identical to the template nucleicacid. In some embodiments, the blocked primers are universal forwardand/or reverse primers, i.e., complementary or identical to multipledifferent templates that comprise different sequences. In someembodiments, the forward or the reverse primer is blocked. In someembodiments, both the forward and reverse primers are blocked.

In some embodiments, the one or more blocked primers comprise a“forward” primer and a “reverse” primer. Placing both primers and thetemplate in contact optionally results in a first double strandedstructure at a first portion of said first strand and a double strandedstructure at a second portion of said second strand. Optionally, the 3′end of the forward and/or reverse primer (after cleavage by RNase Henzyme) is extended with one or more polymerases to generate a first andsecond double stranded nucleic acid and a first and second displacedstrand of nucleic acid. Optionally, the second displaced strand is atleast partially complementary to each other and can hybridize to form adaughter double stranded nucleic acid which can serve as double strandedtemplate nucleic acid in a subsequent amplification cycles.

In some embodiments, In some embodiments, a blocked primer, forwardprimer and/or reverse primer, used in any of the methods provided hereinincludes a 5′ domain and a 3′ domain separated by at least onenucleotide comprising a ribobase. In some embodiments, the 5′ domain and3′ domain are separated by a single ribobase. In some embodiments, the5′ domain and 3′ domain are separated by consecutive ribobases, such astwo, three, four, five or more ribobases. For example, the 5′ domain and3′ domain can be separated by between 1 and 5 consecutive ribobases. Insome embodiments, the ribobases are rU, rG, rA, or rC.

In some embodiments, the 3′ domain of the blocked primers contain ablocking moiety, which is removed after cleavage at the ribobaselocation, once hybridized to the DNA template, with an RNase H enzyme.The block, or blocking group, is a chemical moiety on the end of the 3′primer and prevents primer extension, effectively blocking nucleic acidamplification. Once the blocking group is removed, the hybridized 5′domain of the primer is capable of participating in primer extension andRPA nucleic acid amplification. In some embodiments, the blocking groupcan be any moiety that prevents or blocks primer extension. In someembodiments, the block is a C3 spacer, a phosphate, biotin, or aminemoiety.

In some embodiments, the 5′ domain of the blocked primer can be anylength, but is typically at least 10 nucleotides in length. In someembodiments, the 5′ domain is between 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90 nucleotides on thelow end of the range, and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides on the high endof the range. In some embodiments, the 5′ domain is typically at least15 nucleotides in length and accordingly, in some embodiments is between15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 55, 60, 70, 80 or 90 nucleotides on the low end of the range,and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 55, 60, 70, 80, 90, or 100 nucleotides on the high end of therange. In some embodiments, the 5′ domain can be at least 25 nucleotidesin length and accordingly, in some embodiments the 5′ domain is between25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50,55, 60, 65, 70, 80 or 90 nucleotides on the low end of the range, and26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55,60, 65, 70, 80, 90, or 100 nucleotides on the high end of the range. Insome embodiments, the 5′ domain is 30 nucleotides in length.

In some embodiments, the 5′ domain can be between 10 and 60 nucleotidesin length, and accordingly In some embodiments, the 5′ domain is between10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or 55nucleotides on the low end of the range, and 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or 60 nucleotides on the high endof the range. In some embodiments, the 5′ domain can be between 15 and60 nucleotides in length, and accordingly in some embodiments, the 5′domain is between 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50 or 55 nucleotides on the low end of the range, and 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55 or 60 nucleotideson the high end of the range. In some embodiments, the 5′ domain can bebetween 25 and 60 nucleotides in length, and accordingly In someembodiments, the 5′ domain is between 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 34, 35, 40, 45, 50 or 55 nucleotides on the low end of therange, and 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55 or 60nucleotides on the high end of the range. In some embodiments, the 5′domain can be between 10 and 40 nucleotides in length, and accordinglyIn some embodiments, the 5′ domain is between 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30 or 35 nucleotides on the low end of therange, and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35 or 40 nucleotides on the high end of the range. In someembodiments, the 5′ domain can be between 15 and 40 nucleotides inlength, and accordingly in some embodiments, the 5′ domain is between15, 16, 17, 18, 19, 20, 25, 30 or 35 nucleotides on the low end of therange, and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40nucleotides on the high end of the range. In some embodiments, the 5′domain can be between 25 and 40 nucleotides in length, and accordinglyin some embodiments, the 5′ domain is 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

In some embodiments, the 5′ domain can be between 10 and 30 nucleotidesin length, and accordingly in in some embodiments, the 5′ domain isbetween 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28 or 29 nucleotides on the low end of the range, and 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 30, nucleotides onthe high end of the range. In some embodiments, the 5′ domain can bebetween 15 and 30 nucleotides in length, and accordingly in someembodiments, the 5′ domain is between 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28 or 29 nucleotides on the low end of the range,and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 30, nucleotides on thehigh end of the range.

In some embodiments, the blocked primers comprise a 5′ domain that has alength of at least 10 nucleotides, at least 15 nucleotides, at least 25nucleotides or 30 or more nucleotides. In some embodiments, the blockedprimers comprise a 5′ domain with a range of nucleotide lengths from 10to 100, 10 to 60, 10 to 50, 10 to 40, 10 to 30 or 10 to 25. In someembodiments, the blocked primers comprise a 5′ domain with a range ofnucleotide lengths from 15 to 100, 15 to 60, 15 to 50, 15 to 40, 15 to30 or 15 to 25. In some embodiments, the blocked primers comprise a 5′domain with a range of nucleotide lengths from 25 to 100, 25 to 60, 25to 50, 25 to 40 or 25 to 30.

In some embodiments, the 5′ domain can be between 15 and 25 nucleotidesin length. See V5 primer configuration of FIG. 2, FIG. 10, Table 2 ofExample 1 and corresponding FIGS. 4 and 5. In some embodiments, the 5′domain can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotidesin length. In some embodiments, the 5′ domain can be 15 or 17nucleotides in length. In some embodiments, the 5′ domain of the blockedprimers can be at least 25 nucleotides in length. See V2 primerconfiguration of FIG. 2, FIG. 10, Table 1 of Example 1 and correspondingFIG. 3. In some embodiments, the 5′ domain can be between 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 or 55nucleotides on the low end of the range, and 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 70, 75, or 80nucleotides on the high end of the range. In some embodiments, the 5′domain can be between 30, 31, 32, 33 or 34 nucleotides on the low end ofthe range, and 31, 32, 33, 34 or 35 nucleotides on the high end of therange.

In some embodiments, the 3′ domain is at least 10 nucleotides in length,but can be 7, 8 or 9 nucleotides in length. In some embodiments, the 3′domain is not less than 10 nucleotides in length and in some embodimentsthe 3′ domain is not less 6 nucleotides in length. In some embodiments,the 3′ domain is between 10 and 30 nucleotides in length, wherein the 3′domain is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the3′ domain is typically at least 14 nucleotides in length. In someembodiments, the 3′ domain is between 14 and 30 nucleotides in length,wherein the 3′ domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the 3′domain of the blocked primers is between 15 and 30 nucleotides inlength, and accordingly In some embodiments, the 3′ domain is 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides inlength. In both the V2 and V5 primer configuration illustrated in FIG. 2and FIG. 10; and in the Examples section herein, the 3′ domain was atleast 14 nucleotides in length.

In some embodiments, the 3′ domain is between 10 and 25 nucleotides inlength, and accordingly in some embodiments, the 3′ domain is 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides inlength. In some embodiments, the 3′ domain is between 14 and 25nucleotides in length, and accordingly in some embodiments, the 3′domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotidesin length. In some embodiments, the 3′ domain of the blocked primers isbetween 15 and 25 nucleotides in length, and accordingly in someembodiments, the 3′ domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or25 nucleotides in length. In some embodiments, the 3′ domain of theblocked primers is between 14 and 20 nucleotides in length, andaccordingly in some embodiments, the 3′ domain is 14, 15, 16, 17, 18, 19or 20 nucleotides in length. In some embodiments, the 3′ domain of theblocked primers is between 15 and 20 nucleotides in length, andaccordingly in some embodiments, the 3′ domain is 15, 16, 17, 18, 19 or20 nucleotides in length.

In some embodiments, the blocked primers comprise a 5′ domain and a 3′domain with a length as disclosed herein. In some embodiments, theblocked primers comprise a 5′ domain with a length between 10 and 100nucleotides and a 3′ domain with a length between 10 and 30 nucleotides,and accordingly in some embodiments, the 5′ domain is between 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80or 90 nucleotides on the low end of the range, and 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100nucleotides on the high end of the range and the 3′ domain is between 10and 30 nucleotides, and accordingly in some embodiments, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length. In some embodiments, the blocked primers comprisea 5′ domain with a length between 10 and 100 nucleotides and a 3′ domainwith a length between 14 and 30 nucleotides, and accordingly in someembodiments, the 5′ domain is between 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90 nucleotides on thelow end of the range, and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides on the high endof the range and the 3′ domain is between 14 and 30, and accordingly insome embodiments, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30 nucleotides in length. In some embodiments, the blockedprimers comprise a 5′ domain with a length between 10 and 100nucleotides and a 3′ domain with a length between 15 and 30 nucleotides,and accordingly in some embodiments, the 5′ domain is between 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80or 90 nucleotides on the low end of the range, and 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100nucleotides on the high end of the range and the 3′ domain is between 15and 30 nucleotides in length, and accordingly in some embodiments, the3′ domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 onthe low end of the range, and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 nucleotides in length on the high end of the range.In some embodiments, the blocked primers comprise a 5′ domain with alength of 30 nucleotides and a 3′ domain with a length of 15nucleotides.

In some embodiments, the blocked primers comprise a 5′ domain with alength between 15 and 60 nucleotides and a 3′ domain with a lengthbetween 10 and 30 nucleotides, wherein the 5′ domain is between 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or 55 nucleotideson the low end of the range, and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55 or 60 nucleotides on the high end of the rangeand the 3′ domain is 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 nucleotides on the low end of the range, and 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in lengthon the high end of the range. In some embodiments, the blocked primerscomprise a 5′ domain with a length between 15 and 60 nucleotides and a3′ domain with a length between 14 and 30 nucleotides, wherein the 5′domain is between 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50 or 55 nucleotides on the low end of the range, and 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55 or 60 nucleotideson the high end of the range and the 3′ domain is 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.In some embodiments, the blocked primers comprise a 5′ domain with alength between 15 and 60 nucleotides and a 3′ domain with a lengthbetween 15 and 30 nucleotides, wherein the 5′ domain is between 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or 55 nucleotideson the low end of the range, and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55 or 60 nucleotides on the high end of the rangeand the 3′ domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 nucleotides in length.

In some embodiments, the blocked primers comprise a 5′ domain with alength between 25 and 60 nucleotides in length and a 3′ domain with alength between 10 and 30 nucleotides, wherein the 5′ domain is between25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 40, 45, 50 or 55nucleotides on the low end of the range, and 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 40, 45, 50, 55 or 60 nucleotides on the high end of therange and the 3′ domain is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In someembodiments, the blocked primers comprise a 5′ domain with a lengthbetween 25 and 60 nucleotides in length and a 3′ domain with a lengthbetween 14 and 30 nucleotides, wherein the 5′ domain is between 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 40, 45, 50 or 55 nucleotides onthe low end of the range, and 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,40, 45, 50, 55 or 60 nucleotides on the high end of the range and the 3′domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 nucleotides in length. In some embodiments, the blocked primerscomprise a 5′ domain with a length between 25 and 60 nucleotides inlength and a 3′ domain with a length between 15 and 30 nucleotides,wherein the 5′ domain is between 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,34, 35, 40, 45, 50 or 55 nucleotides on the low end of the range, and26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55 or 60 nucleotideson the high end of the range and the 3′ domain is 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

In some embodiments, the blocked primers comprise a 5′ domain with alength between 15 and 30 nucleotides and a 3′ domain with a lengthbetween 14 and 30 nucleotides, wherein the 5′ domain is 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in lengthand the 3′ domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30 nucleotides in length. In some embodiments, the blockedprimers comprise a 5′ domain that is 30 nucleotides in length and a 3′domain that is 15 nucleotides in length. See Example 2.

In some embodiments, the blocked primers comprise a 5′ domain with alength of 24 or 25 nucleotides, a ribobase such as rA or rG, a 3′ domainwith a length of 5 or 6 nucleotides and a blocking moiety. See FIG. 10and V1 primer configuration. In some embodiments, the blocked primerscomprise a 5′ domain with a length of 30, 31, 32, 33, 34 or 35nucleotides, a ribobase such as rC or rU, a 3′ domain with a length of15, 16 or 17 nucleotides and a blocking moiety. See FIG. 10 and V2primer configuration. In some embodiments, the blocking primers comprisea 5′ domain with a length of 31 or 35 nucleotides, a ribobase such asrC, a 3′ domain with a length of 5 nucleotides and a blocking moiety.See FIG. 10 and V3 primer configuration. In some embodiments, theblocked primers comprise a 5′ domain with a length of 30 or 32nucleotides, a ribobase such as rU, a 3′ domain with a length of 10nucleotides and a blocking moiety. See FIG. 10 and V4 primerconfiguration. In some embodiments, the blocked primers comprise a 5′domain with a length of 15 or 17 nucleotides, a ribobase such as rG orrU, a 3′ domain with a length of 15 nucleotides and a blocking moiety.See FIG. 10 and V5 primer configuration.

In some embodiments, the blocked primers comprise a 5′ domain that is atleast 25 nucleotides and a 3′ domain that is at least 14 nucleotides inlength, wherein the 5′ domain is between 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80 or 90nucleotides on the low end of the range, and 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 100nucleotides on the high end of the range and the 3′ domain is 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotidesin length. See primer configuration V2 of Example 1. In someembodiments, the blocked primers comprise a 5′ domain that is 15 to 25nucleotides and a 3′ domain that is at least 14 nucleotides in length,wherein the 5′ domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25nucleotides in length and the 3′ domain is 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. Seeprimer configuration V5 of Example 1.

In some embodiments, the blocked primers comprise a 5′ domain that is atleast 25 nucleotides wherein, the 3′ domain is 14 nucleotides; or the 3′domain is 15 nucleotides; or the 3′ domain is 16 nucleotides; or the 3′domain is 17 nucleotides; or the 3′ domain is 18 nucleotides; or the 3′domain is 19 nucleotides; or the 3′ domain is 20 nucleotides; or the 3′domain is 21 nucleotides; or the 3′ domain is 22 nucleotides; or the 3′domain is 23 nucleotides; or the 3′ domain is 24 nucleotides; or the 3′domain is 25 nucleotides; or the 3′ domain is 26 nucleotides; or the 3′domain is 27 nucleotides; or the 3′ domain is 28 nucleotides; or the 3′domain is 29 nucleotides; or the 3′ domain is 30 nucleotides. In someembodiments, the blocked primers comprise a 5′ domain that is between 15and 25 nucleotides in length, wherein, the 3′ domain is 14 nucleotides;or the 3′ domain is 15 nucleotides; or the 3′ domain is 16 nucleotides;or the 3′ domain is 17 nucleotides; or the 3′ domain is 18 nucleotides;or the 3′ domain is 19 nucleotides; or the 3′ domain is 20 nucleotides;or the 3′ domain is 21 nucleotides; or the 3′ domain is 22 nucleotides;or the 3′ domain is 23 nucleotides; or the 3′ domain is 24 nucleotides;or the 3′ domain is 25 nucleotides; or the 3′ domain is 26 nucleotides;or the 3′ domain is 27 nucleotides; or the 3′ domain is 28 nucleotides;or the 3′ domain is 29 nucleotides; or the 3′ domain is 30 nucleotides.

In some embodiments, the blocked primers comprise a 5′ domain that is 10to 100 nucleotides in length and a 3′ domain that is 11 to 30nucleotides in length. In some embodiments, the 5′ domain is 15 to 50nucleotides in length. In some embodiments, the 5′ domain is 15 to 30nucleotides. In some embodiments, the 3′ domain is 14 to 25 nucleotidesin length or 15 to 25 nucleotides length. In some embodiments, the 3′domain is 14 to 20 nucleotides in length wherein the ribobase is rU, rGor rA. In some embodiments, the 3′ domain is 15 to 20 nucleotides inlength wherein the ribobase is rU, rG or rA.

In some embodiments, the 3′ domain optionally comprises a mismatchedbase pair. In some embodiments, a 3′ nucleotide of the 3′ domain of ablocked forward primer is mismatched to a forward primer bindingsequence. In some embodiments, a 3′ nucleotide of the 3′ domain of ablocked reverse primer is mismatched to a reverse primer bindingsequence. In some embodiments, the 3′ domain optionally comprises morethan one mismatched base pair.

In some embodiments, the methods, compositions and kits described hereinfor amplifying nucleic acid template, at least one blocked primer isused wherein the 3′ domain is from 10 to 30 nucleotides in length. Insome embodiments, the compositions and kits comprise at least oneblocked primer wherein the 3′ domain is from 10 to 30 nucleotides inlength.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits for amplifying nucleic acids, astandard (non-blocked) primer can be used in combination with at leastone blocked primer. In some embodiments, the non-blocked primerstypically have a free 3′ hydroxyl. It is understood that use of the term“standard primer” refers to a non-blocked primer (no ribobase cleavagelocation or blocking moiety) and not a blocked primer of the invention.In some embodiments, standard primers comprise polymers ofdeoxyribonucleotides or analogs thereof. In some embodiments, standardprimers comprise naturally-occurring, synthetic, recombinant, cloned,amplified, or unamplified forms. In some embodiments, non-blockedprimers include phosphodiester linkages between all nucleotides.

In some embodiments, standard primers can be any length, including about5-100 nucleotides, or about 10-100 nucleotides, or about 15-100nucleotides, or about 20-100 nucleotides, or longer.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits for amplifying nucleic acids,wherein in addition to the blocked primers discussed above, a reactionmixture is formed containing the necessary components for amplificationof the template nucleic acid. Those components, in some embodiments,include one or more nucleic acid templates, a polymerase, a recombinase,a recombinase accessory protein, a forward primer, a reverse primer,dNTPs, an RNase H enzyme, a buffer, and optionally a sieving agent, andoptionally a crowding agent, and optionally a single-stranded bindingprotein.

In some embodiments, methods for nucleic acid amplification can includeat least one co-factor for recombinase or polymerase activity. In someembodiments, a co-factor comprises one or more divalent cation. Examplesof divalent cations include magnesium, manganese and calcium. In someembodiments, the reaction mixture comprises a buffer comprising adivalent cation. In embodiments the buffer comprises magnesium ormanganese ions.

In some embodiments, the reaction mixture may be formed by theindividual addition of each component to an aqueous or emulsionsolution. In some embodiments, the reaction mixture can be in the formof a dehydrated pellet that requires rehydration prior to use. In someembodiments, the reaction mixture is in the form of a dehydrated pelletand comprises recombinase, recombinase accessory proteins, gp32, DNApolymerase, dNTPs, ATP, phosphocreatine, a crowding agent and creatinekinase. See Example 1. Rehydration buffer can include, for example, Trisbuffer, potassium acetate salt and a crowding agent such as PEG.

In some embodiments, the reaction mixture is in the form of a dehydratedpellet and comprises recombinase, recombinase accessory protein(s),gp32, T7 DNA polymerase, thioredoxin, dNTPs, ATP, phosphocreatine, acrowding agent and creatine kinase. In some embodiments, when adehydrated pellet is used that includes reaction mixture components, thepellet is rehydrated with a rehydration buffer, template DNA, primersincluding blocked primers of the invention, RNase H enzyme andadditional nuclease-free water are added to a final volume.

In some embodiments, a nucleic acid amplification reaction can bepre-incubated under conditions that inhibit premature reactioninitiation. For example, one or more components of a nucleic acidamplification reaction can be withheld from a reaction vessel to preventpremature reaction initiation. To start the reaction, a divalent cationcan be added (e.g., magnesium or manganese). In another example, anucleic acid amplification reaction can be pre-incubated at atemperature that inhibits enzyme activity. The reaction can bepre-incubated at about 0-15° C., or about 15-25° C. to inhibit prematurereaction initiation. The reaction can then be incubated at a highertemperature to induce enzymatic activity. In some embodiments, thereaction mixture is not exposed to a temperature above 40° C. during theamplification. Further details and examples of reaction mixtures andcomponents thereof, are found herein, for example in discussions ofcomposition embodiments as well as discussion herein related toindividual components of the reaction mixtures and compositions.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits for amplifying nucleic acids,wherein the nucleic acid templates (e.g., nucleic acid templates)include a forward primer binding site having a forward primer bindingsequence and a reverse primer binding site having a reverse primerbinding sequence. In some embodiments, the primers are referred to as afirst and a second primer wherein the template comprises a first primerbinding sequence and a second primer sequence. In embodiments the firstand second primers are blocked universal primers, such as 3′ blockeduniversal primers that are cleavable by RNase H. In some embodiments,the reaction mixture comprises one monoclonal template nucleic acid. Insome embodiments, reaction mixture comprises at least two different(polyclonal) polynucleotide or nucleic acid templates.

In some embodiments, the reaction mixture for the methods for nucleicacid amplification comprise a plurality of different polynucleotides. Insome embodiments, a plurality of different polynucleotides comprisessingle-stranded or double-stranded polynucleotides, or a mixture ofboth. In some embodiments, a plurality of different polynucleotidescomprises polynucleotides having the same or different sequences. Insome embodiments, a plurality of different polynucleotides comprisespolynucleotides having the same or different lengths. In someembodiments, a plurality of different polynucleotides comprises about2-10, or about 10-50, or about 50-100, or about 100-500, or about500-1,000, or about 1,000-5,000, or about 10³-10⁶, or about 10⁶-10¹⁰ ormore different polynucleotides. In some embodiments, a plurality ofdifferent polynucleotides comprises polymers of deoxyribonucleotides,ribonucleotides, and/or analogs thereof. In some embodiments, aplurality of different polynucleotides comprises naturally-occurring,synthetic, recombinant, cloned, amplified, unamplified or archived(e.g., preserved) forms. In some embodiments, a plurality of differentpolynucleotides comprises DNA, cDNA RNA or chimeric RNA/DNA, and nucleicacid analogs.

In some embodiments, a plurality of different polynucleotide templatesamplified in methods provided herein can comprise a double-strandedpolynucleotide library construct having a nucleic acid adaptor sequenceon one or both ends. For example, a polynucleotide library construct cancomprise a first and second end, where the first end is joined to afirst nucleic acid adaptor. A polynucleotide library construct can alsoinclude a second end joined to a second nucleic acid adaptor. The firstand second adaptors can have the same or different sequence. In someembodiments, at least a portion of the first or second nucleic acidadaptor (i.e., as part of the polynucleotide library construct) canhybridize to the first primer, which can be a universal primer. In someembodiments, a homologous recombination enzyme, as part of anucleoprotein complex, can bind to a polynucleotide library constructhaving a first or second nucleic acid adaptor sequence.

In some embodiments, polynucleotide library constructs can be compatiblefor use in any type of sequencing platform including chemicaldegradation, chain-termination, sequence-by-synthesis, pyrophosphate,massively parallel, ion-sensitive, single molecule platforms, andcombinations thereof.

In some embodiments, methods for nucleic acid amplification includediluting the amount of polynucleotides that are reacted with beads(e.g., beads attached with a plurality of a first primer, such as afirst RNase cleavable blocked primer of the invention) to reduce thepercentage of beads that react with more than one polynucleotide. Insome embodiments, nucleic acid amplification reactions can be conductedwith a polynucleotide-to-bead ratio that is selected to optimize thepercentage of beads having a monoclonal population of polynucleotidesattached thereto. For example, a nucleic acid amplification reaction canbe conducted at anyone of polynucleotide-to-bead ratios in a range ofabout 1:1 or 1:2 to 1:500. In some embodiments, a polynucleotide-to-beadratio includes about 1:1, or about 1:2, or about 1:5, or about 1:10, orabout 1:25, or about 1:50, or about 1:75, or about 1:100, or about1:125, or about 1:150, or about 1:175, or about 1:200, or about 1:225,or about 1:225, or about 1:250. In some embodiments, a nucleic acidamplification reaction can produce beads having zero types ofpolynucleotides attached thereto, other beads having one type ofpolynucleotide attached thereto, and other beads having more than onetype of polynucleotides attached thereto.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits for amplifying nucleic acids,wherein the reaction mixtures comprise a recombinase. Similarly,compositions and kits provided herein can include a recombinase. Therecombinase can include any agent that is capable of inducing, orincreasing the frequency of occurrence, of a recombination event. Arecombination event includes any event whereby two differentpolynucleotides strands are recombined with each other. Recombinationcan include homologous recombination. The recombinase can be an enzyme,or a genetically engineered derivative thereof. The recombinaseoptionally can associate with (e.g., bind) a single-strandoligonucleotide (e.g., a first primer). In some embodiments, an enzymethat catalyzes homologous recombination can form a nucleoprotein complexby binding a single-stranded oligonucleotide. In some embodiments, ahomologous recombination enzyme, as part of a nucleoprotein complex, canbind a homologous portion of a double-stranded polynucleotide. In someembodiments, the homologous portion of the polynucleotide can hybridizeto at least a portion of the first primer. In some embodiment, thehomologous portion of the polynucleotide can be partially or completelycomplementary to at least a portion of the first primer.

In some embodiments, a homologous recombination enzyme can catalyzestrand invasion by forming a nucleoprotein complex and binding to ahomologous portion of a double-stranded polynucleotide to form arecombination intermediate having a triple-strand structure (D-loopformation) (U.S. Pat. No. 5,223,414 to Zarling, U.S. Pat. Nos. 5,273,881and 5,670,316 both to Sena, and U.S. Pat. Nos. 7,270,981, 7,399,590,7,435,561, 7,666,598, 7,763,427, 8,017,339, 8,030,000, 8,062,850, and8,071,308).

In some embodiments, the recombinase of the reaction mixtures,compositions, and kits provided herein can include any suitable agentthat can promote recombination between polynucleotide molecules. Therecombinase can be an enzyme that catalyzes homologous recombination.For example, the reaction mixture can include a recombinase thatincludes, or is derived from, a bacterial, eukaryotic or viral (e.g.,phage) recombinase enzyme.

In some embodiments, a homologous recombination enzyme compriseswild-type, mutant, recombinant, fusion, or fragments thereof. In someembodiments, a homologous recombination enzyme comprises an enzyme fromany organism, including myoviridae (e.g., uvsX from bacteriophage T4,RB69, and the like) Escherichia coli (e.g., recA), or human (e.g.,RAD51). In some embodiments, the reaction mixture includes one or morerecombinases selected from uvsX, RecA, RadA, RadB, Rad51, a homologuethereof, a functional analog thereof or a combination thereof. Therecombinase in illustrative examples is uvsX. The UvsX protein can bepresent, for example, at 50-250 ng/ul or 100-200 ng/ul.

In some embodiments, methods for nucleic acid amplification comprise oneor more accessory proteins. For example, an accessory protein canimprove the activity of a recombinase enzyme (U.S. Pat. No. 8,071,308granted to Piepenburg, et al.). In some embodiments, an accessoryprotein can bind single strands of nucleic acids, or can load arecombinase onto a nucleic acid. In some embodiments, an accessoryprotein comprises wild-type, mutant, recombinant, fusion, or fragmentsthereof. In some embodiments, accessory proteins can originate from anycombination of the same or different species as the recombinase enzymethat are used to conduct a nucleic acid amplification reaction.Accessory proteins can originate from any bacteriophage including amyoviral phage. Examples of a myoviral phage include T4, T2, T6, Rb69,Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophageP-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage25, Vibrio phage nt-1, phi-1, Rb16, Rb43, Phage 31, phage 44RR2.8t,Rb49, phage Rb3, and phage LZ2. Accessory proteins can originate fromany bacterial species, including Escherichia coli, Sulfolobus (e.g., S.solfataricus) or Methanococcus (e.g., M. jannaschii).

In some embodiments, methods for nucleic acid amplification can includesingle-stranded binding proteins. Single-stranded binding proteinsinclude myoviral gp32 (e.g., T4 or RB69), Sso SSB from Sulfolobussolfataricus, MjA SSB from Methanococcus jannaschii, or E. coli SSBprotein.

In some embodiments, methods for nucleic acid amplification can includeproteins that can improve recombinase loading onto a nucleic acid. Forexample, a recombinase loading protein comprises a UvsY protein (U.S.Pat. No. 8,071,308 granted to Piepenburg). In some embodiments, thereaction mixture includes recombinase accessory proteins. In someembodiments, the recombinase accessory protein is uvsY. UvsY can bepresent, for example, at 20 ng/ul to 100 ng/ul.

In some embodiments, the reaction mixture used herein for nucleic acidamplification may include at least one co-factor for recombinaseassembly on nucleic acids or for homologous nucleic acid pairing. Insome embodiments, a co-factor comprises any form of ATP including ATPand ATPγS.

In some embodiments, methods for nucleic acid amplification can includeat least one co-factor that regenerates ATP. For example, a co-factorcomprises an enzyme system that converts ADP to ATP. In someembodiments, a co-factor comprises phosphocreatine and creatine kinase.

The reaction mixture further comprises nucleotides (dNTPs) for strandextension of one or more nucleic acid templates, and in some embodimentsresulting in a clonal population of the template nucleic acid sequence.In some embodiments, the nucleotides are not extrinsically labeled. Forexample, the nucleotides can be naturally occurring nucleotides, orsynthetic analogs that do not include fluorescent moieties, dyes, orother extrinsic optically detectable labels. Optionally, the reactionmixture includes nucleotides that are naturally occurring nucleotides.Optionally, the nucleotides do not include groups that terminate nucleicacid synthesis (e.g., dideoxy groups, reversible terminators, and thelike).

Optionally, the reaction mixture includes nucleotides that are naturallyoccurring nucleotides. Optionally, the nucleotides do not include groupsthat terminate nucleic acid synthesis (e.g., dideoxy groups, reversibleterminators, and the like). In some embodiments, the nucleotidescomprise a label or tag, described in more detail below.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits for nucleic acid amplificationwhich include contacting (e.g., mixing) one or more nucleic acidtemplates with one or more primers in the presence of one or moreenzymes capable of polymerization. In some embodiments, the one or moreenzymes capable of polymerization include at least one polymerase and arecombinase. In some embodiments, the at least one polymerase includes athermostable or thermolabile polymerase. In some embodiments, the atleast one polymerase includes a biologically active fragment of a DNA orRNA polymerase that maintains sufficient catalytic activity topolymerize or incorporate at least one nucleotide under any suitableconditions. In one embodiment, the at least one polymerase comprises amutated DNA or RNA polymerase that maintains sufficient catalyticactivity to perform nucleotide polymerization under any suitableconditions. In another embodiment, the at least one polymerase includesone or more amino acid mutations that do not disrupt processivity of thepolymerase; and wherein the at least one polymerase having at least onemutation maintains sufficient catalytic activity to performpolymerization.

In some embodiments, a polymerase includes any enzyme, or fragment orsubunit of thereof, that can catalyze polymerization of nucleotidesand/or nucleotide analogs. In some embodiments, a polymerase requires anextendible 3′ end. For example, a polymerase requires a terminal 3′ OHof a nucleic acid primer to initiate nucleotide polymerization. Thepolymerase can be other than a thermostable polymerase. For example, thepolymerase can be active at 37° C. and/or more active at 37° C. than at50° C., 60° C., 70° C. or higher.

In some embodiments, a polymerase comprises any enzyme that can catalyzethe polymerization of nucleotides (including analogs thereof) into anucleic acid strand. Typically, but not necessarily such nucleotidepolymerization can occur in a template-dependent fashion. In someembodiments, a polymerase can be a high fidelity polymerase. Suchpolymerases can include without limitation naturally occurringpolymerases and any subunits and truncations thereof, mutantpolymerases, variant polymerases, recombinant, fusion or otherwiseengineered polymerases, chemically modified polymerases, syntheticmolecules or assemblies, and any analogs, derivatives or fragmentsthereof that retain the ability to catalyze such polymerization.Optionally, the polymerase can be a mutant polymerase comprising one ormore mutations involving the replacement of one or more amino acids withother amino acids, the insertion or deletion of one or more amino acidsfrom the polymerase, or the linkage of parts of two or more polymerases.The term “polymerase” and its variants, as used herein, also refers tofusion proteins comprising at least two portions linked to each other,where the first portion comprises a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand and is linkedto a second portion that comprises a second polypeptide, such as, forexample, a reporter enzyme or a processivity-enhancing domain.Typically, the polymerase comprises one or more active sites at whichnucleotide binding and/or catalysis of nucleotide polymerization canoccur. In some embodiments, a polymerase includes or lacks otherenzymatic activities, such as for example, 3′ to 5′ exonuclease activityor 5′ to 3′ exonuclease activity. In some embodiments, a polymerase canbe isolated from a cell, or generated using recombinant DNA technologyor chemical synthesis methods. In some embodiments, a polymerase can beexpressed in prokaryote, eukaryote, viral, or phage organisms. In someembodiments, a polymerase can be post-translationally modified proteinsor fragments thereof.

In some embodiments, the polymerase can include any one or morepolymerases, or biologically active fragment of a polymerase, which isdescribed in U.S. Patent Publ. No. 2011/0262903 to Davidson et al.,published Oct. 27, 2011, and/or International PCT Publ. No. WO2013/023176 to Vander Horn et al., published Feb. 14, 2013.

In some embodiments, a polymerase can be a DNA polymerase and includewithout limitation bacterial DNA polymerases, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases.

In some embodiments, a polymerase can be a replicase, DNA-dependentpolymerase, primases, RNA-dependent polymerase (including RNA-dependentDNA polymerases such as, for example, reverse transcriptases), athermo-labile polymerase, or a thermo-stable polymerase. In someembodiments, a polymerase can be any Family A or B type polymerase. Manytypes of Family A (e.g., E. coli Pol I), B (e.g., E. coli Pol II), C(e.g., E. coli Pol III), D (e.g., Euryarchaeotic Pol II), X (e.g., humanPol beta), and Y (e.g., E. coli UmuC/DinB and eukaryotic RAD30/xerodermapigmentosum variants) polymerases are described in Rothwell and Watsman2005 Advances in Protein Chemistry 71:401-440. In some embodiments, apolymerase can be a T3, T5, T7, or SP6 RNA polymerase.

In some embodiments, nucleic acid amplification reactions can beconducted with one type or a mixture of polymerases, recombinases and/orligases. In some embodiments, nucleic acid amplification reactions canbe conducted with a low fidelity or high fidelity polymerase.

In some embodiments, the reaction mixture can include a polymerase. Thepolymerase optionally has, or lacks, exonuclease activity. In someembodiments, the polymerase has 5′ to 3′ exonuclease activity, 3′ to 5′exonuclease activity, or both. Optionally, the polymerase lacks any oneor more of such exonuclease activities.

In some embodiments, the polymerase has strand displacing activity.Examples of useful strand-displacing polymerases include Bacteriophage029 DNA polymerase and Bst DNA polymerase.

An exemplary polymerase is Bst DNA Polymerase (Exonuclease Minus), is a67 kDa Bacillus stearothermophilus DNA Polymerase protein (largefragment), exemplified in accession number 2BDP_A, which has 5′-3′polymerase activity and strand displacement activity but lacks 3′-5′exonuclease activity. Other polymerases include Taq DNA polymerase Ifrom Thermus aquaticus (exemplified by accession number 1TAQ), Eco DNApolymerase I from Escherichia coli (accession number P00582), Aea DNApolymerase I from Aquifex aeohcus (accession number 067779), orfunctional fragments or variants thereof, e.g., with at least 80%, 85%,90%, 95% or 99% sequence identity at the nucleotide level.

In some embodiments, the DNA polymerase is a Bsu DNA polymerase (largefragment (NEB). Bsu DNA Polymerase I, Large Fragment retains the 5′→3′polymerase activity of the Bacillus subtilis DNA polymerase I (1), butlacks the 5′→3′ exonuclease domain. In some embodiments, the Bsu DNAPolymerase large fragment lacks 3′→5′ exonuclease activity. In someembodiments, Bsu DNA Polymerase large fragment has optimal activity at37° C.

In one embodiment, the one or more enzymes capable of polymerizationinclude a T5 or T7 DNA polymerase. In some embodiments, the one or moreenzymes capable of polymerization include a T5 or T7 DNA polymerasehaving one or more amino acid mutations that reduce 3′-5′ exonucleaseactivity. In some embodiments, the T5 or T7 DNA polymerase having one ormore amino acid mutations that reduce 3′-5′ exonuclease activity, doesnot contain an amino acid mutation that disrupts processivity of the T5or T7 DNA polymerase. In some embodiments, the T5 or T7 DNA polymerasecan include one or more amino acid mutations that eliminate detectable3′-5′ exonuclease activity; and wherein the one or more amino acidmutations do not disrupt processivity of the T5 or T7 DNA polymerase. Insome embodiments, the reaction mixture comprises a Sau polymerase, T7DNA polymerase with reduced 3′ to 5′ exonuclease activity, Bsupolymerase, or a combination thereof.

In some embodiments, the one or more enzymes capable of polymerizationcan include any suitable RNA polymerase. Suitable RNA polymerasesinclude, without limitation, T3, T5, T7, and SP6 RNA polymerases.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits, wherein the nucleic acidamplification includes a combination of recombinase-polymeraseamplification (RPA) and the blocked primers under isothermal conditions.During amplification, the blocked primers are cleaved at the ribobase bya ribonuclease (RNase) to permit primer extension and templateamplification. As is known, RNases are enzymes catalyzing hydrolysis ofRNA into smaller components. The use of RNase H enzyme with the blockedprimers of the invention provides advantages over methods of the artwherein their use reduces: 1) nonspecific primer tailing via blocked 3′end; and 2) non-templated primer dimer and nonspecific productformation, via specific RNase H cleavage on RNA/RNA duplex betweenprimer and template strands.

In some embodiments, the compositions (e.g. reaction mixtures), methods,and kits, include a ribo-endonuclease that is active at appropriatetemperatures for recombinase and polymerase activity and are compatiblewith those enzymes. The ribo-endonuclease used in the compositions,methods, and kits provided herein, can be an RNase H enzyme, whichrepresents a family of non-sequence specific endonucleases that catalyzethe cleavage of RNA via a hydrolytic mechanism wherein the enzymecleaves the 3′-O—P bond of RNA in a DNA/RNA duplex substrate, providedthat the RNase H enzyme is active at appropriate temperatures forrecombinase and polymerase activity and is compatible with thoseenzymes.

The RNase H enzyme and its family of enzymes include two classes, type 1and type 2 RNase H based on the difference in their amino acid sequence.Type 1 RNases H include prokaryotic and eukaryotic RNases H1 andretroviral RNase H. Type 2 RNases H include prokaryotic and eukaryoticRNases H2 and bacterial RNase H3. These RNases H exist in a monomericform, except for eukaryotic RNases H2, which exist in a heterotrimericform. All of these enzymes share the characteristic that they are ableto cleave the RNA component of an RNA:DNA heteroduplex or within aDNA:DNA duplex containing RNA base(s) within one or both of the strands.The cleaved product yields a free 3′-OH for both classes of RNase H.RNase H1 requires more than a single RNA base within an RNA:DNA duplexfor optimal activity, whereas RNase HII requires only a single RNA basein an RNA:DNA duplex.

In some embodiments, the RNase H enzyme can be any RNase H enzymes,provided that the enzyme retains sufficient activity at appropriatetemperatures for recombinase and polymerase activity and is compatiblewith those enzymes. Therefore, the kits, compositions, and methods caninclude an RNase H enzyme that has higher activity at 37° C. than it hasat least one of the following temperatures: 60° C., 65° C., 70° C., 75°C., or 80° C. For example, an RNase H enzyme used in the kits,compositions, and methods herein can have a higher activity at 37° C.than at 75° C. or have a higher activity at 37° C. than at 70° C. Insome embodiments, the RNase H enzyme cleaves the oligonucleotide moreefficiently at 37° C. than at 60° C. wherein cleavage of the ribobasepresent in the primer occurs at a temperature below 42° C. Accordingly,in illustrative examples of any of the embodiments provided herein, theRNase H enzyme is not a thermostable RNase H enzyme (i.e. the RNase Henzyme is other than a thermostable RNase H enzyme). In someembodiments, the RNase H enzyme that has significant activity at 20 to42° C. The methods, compositions, and kits provided herein can includein illustrative examples, an RNase H enzyme that has significantactivity at 37° C. In some embodiments, the RNase H has sufficientactivity to carry out the claim methods at 37° C.

As indicated, the RNase H enzyme included in the methods, compositions,and kits provided herein can be any RNase H enzyme that is active atappropriate temperatures for recombinase and polymerase activity and iscompatible with those enzymes. In some embodiments, RNase H enzymecomprises RNase H1 (commercially available from NEB, Inc.) or RNase H3.In alternative embodiments, RNase H enzyme does not comprise RNase H1 orRNase H3. An exemplary RNase H enzyme includes E. coli RNase HII(available for example from NEB, Inc. (product M0288)). In someembodiments, the endonuclease can be an RNase HII which cleaves aribobase within a DNA duplex and leaves a 3′ hydroxyl end, andtemperatures at which it retains high activity are compatible with thoseof recombinase and recombinase associated proteins. In some embodiments,the RNase can be E. coli RNase H (available from NEB, Inc., for exampleproduct M0297) (products/m0297-rnase-h), which also cleaves a ribo-basewhen hybridized to DNA and leaves a 3′-hydroxyl end.

In some embodiments, the methods, as well as related compositions andkits include a blocked primer design with 2-5 consecutive ribobases.RNase H2 from Pyrococcus abyssi (P.a.), however, has low activity atroom temperature with optimal activity around 70° C., a temperatureabove the range for the RPA methods. Accordingly, in some embodimentsherein, a higher temperature is used for primer activation than used foramplification. For example, a primer amplification step at between 42°C. and 70° C., 45° C. and 70° C., or 50° C. and 70° C., or 50° C. and65° C., or 60° C. and 70° C. can be performed, before an amplificationat temperatures disclosed for the amplification methods herein, such asbetween 20° C. and 42° C. Some embodiments include performing primeractivation and polymerization at two separate temperatures. RNase H2,such as RNase HII, can be used in such 2-step methods as well, since itis known to retain activity even at high temperatures.

The use of blocked primers are a potentially rate limiting step in theRPA methods (See FIG. 1), because the 3′ domain and block must beremoved before primer extension can proceed. To ensure the PRA reactionproceeds rapidly, in some embodiments an excess (i.e. non-limiting)amount of the RNase enzyme is used. One of skill understands an excesscan be determined empirically, see Example 3 for example. However inembodiments between 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×,14×, and 15× on the low end of the range, and 5×, 6×, 7×, 8×, 9×, 10×,11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20× or 21×, on the high endof the range concentration of RNase H as compared to a minimally excessconcentration, are used in the RPA methods. The exact concentration willdepend on the starting concentration and other amplification parameters.One unit of RNase is defined as the amount of enzyme required to yield afluorescence signal consistent with the nicking of 100 picomol ofsynthetic double-stranded DNA (dsDNA) substrate containing a singleribonucleotide near the quencher of a fluorophore/quencher pair in 30minutes at 37° C. in 1× ThermoPol Buffer (NEB, Inc.). The dsDNAsubstrate can be a 26-mer present at 30 nM in a total reaction volume of150 μl as indicated in the RNase HII product manual for RNase HIIproduct M0288 of NEB, Inc., and as used to determine unit activity orRNase HII by NEB, Inc.

As described in Example 3, the RNase H enzyme concentration used in theRPA amplification reaction can be characterized as a “prohibiting”,“limiting” or “excess” amount. Those concentrations are determinedempirically and may be different for different blocked primerconfigurations, different concentration of starting DNA template ordifferent reaction times or temperature. In some embodiments, an“excess” amount of RNase H enzyme when used with a V2 or V5 primerconfiguration is 20 U or more in a 50 μL reaction volume. In someembodiments, a “limiting” amount of RNase H enzyme is 5-10 U/50 μL, suchas 5, 6, 7, 8, 9, 10 U/50 μL. In some embodiments, a “limiting” amountof RNase H enzyme is less than 20 U/50 μL, such as 19, 18, 17, 16, 15,14, 13, 12, 11, or 10 U/50 μL. An “excess” amount of RNase H enzyme canbe used in any of the embodiments of the invention provided herein. Suchexcess can be, for example, a concentration of equal to or greater than20 U/50 μL (See FIGS. 8A and 8B).

In some embodiments, an E. coli RNase HII enzyme at a concentration from2.5 U to 200 U/50 μL can be used. For example, an E. coli RNase HIIenzyme present in the reaction mixture at a concentration from 5 to 150,10 to 100, or 10 to 50 U/50 μL can be used. In some embodiments, greaterthan 10 U of RNase HII can be used.

In some embodiments, the RNase HII enzyme is present in the reactionmixture at an excess concentration. For example, the RNase HII enzymecan be an E. coli RNase HII enzyme and can be present at a concentrationfrom 20-250 U/50 μL, 20-200 U/50 μL, 20-150 U/50 μL or 20-100 U/50 μL.As a non-limiting, specific example, the RNase H enzyme can be presentin the reaction mixture at a concentration of 20, 25, 30, 40, 50, 75,100, 150, 200, or 250 U/50 μL. The RNase H enzyme in certain examples ofmethods, kits, compositions, and reaction mixtures provided herein, ispresent at 2×, 3×, 4×, 5×, 10×, 20×, 40× or 50× an excessiveconcentration. For example, the RNase H enzyme can be an E. coli RNaseHII enzyme present at 200, 250, 300, 400, 500, 750, 1000, 1500, 2000, or2500 U/50 μL.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits, wherein the nucleic acidamplification includes a reaction mixture which can include a diffusionlimiting agent. The diffusion limiting agent can be any agent that iseffective in preventing or slowing the diffusion of one or more of thepolynucleotide templates and/or one or more of the amplificationreaction products through the amplification reaction mixture.

In some embodiments, the reaction mixture can include a sieving agent.The sieving agent can be any agent that is effective in sieving one ormore polynucleotides present in the amplification reaction mixture, suchas for example amplification reaction products and/or polynucleotidetemplates. In some embodiments, the sieving agent restricts or slows themigration of polynucleotide amplification products through the reactionmixture.

Inclusion of a sieving agent may be advantageous when clonallyamplifying two or more nucleic acid templates within a single continuousliquid phase of a reaction mixture. For example, the sieving agent canprevent or slow diffusion of templates, or amplified polynucleotidesproduced via replication of at least some portion of a template, withinthe reaction mixture, thus preventing the formation of polyclonalcontaminants without requiring compartmentalization of the reactionmixture by physical means or encapsulation means (e.g., emulsions)during the amplification. Such methods of clonally amplifying templateswithin a single continuous liquid phase of a single reaction mixturewithout need for compartmentalization greatly reduces the cost, time andeffort associated with generation of libraries amenable forhigh-throughput methods such as digital PCR, next generation sequencing,and the like.

In some embodiments, the average pore size of the sieving agent is suchthat movement of a target component within the reaction mixture (e.g., apolynucleotide) is selectively retarded or prevented. In one example,the sieving agent comprises any compound that can provide a matrixhaving a plurality of pores that are small enough to slow or retard themovement of a polynucleotide through a reaction mixture containing thesieving agent. Thus, a sieving agent can reduce Brownian motion of apolynucleotide.

In some embodiments, the amplification includes amplifying a pluralityof different polynucleotide templates onto a plurality of different beadsupports in the presence of a sieving agent, and recovering a percentageof substantially monoclonal bead supports, each such substantiallymonoclonal bead support include a bead support attached to asubstantially monoclonal polynucleotide population. In some embodiments,the percentage of substantially monoclonal bead supports recovered issubstantially greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 89%, 90%, or 95% of total amplified beadsupports (i.e., total bead supports including either polyclonal ormonoclonal populations) recovered from the reaction mixture. In someembodiments, the percentage of substantially monoclonal bead supportsrecovered is substantially greater than the percentage of substantiallymonoclonal bead supports recovered following amplification in theabsence of the sieving agent but under otherwise essentially similar orsame amplification conditions.

In some embodiments, a sieving agent comprises a polymer compound. Insome embodiments, a sieving agent comprises a cross-linked or anon-cross linked polymer compounds. By way of non-limiting examples, thesieving agent can include polysaccharides, polypeptides, organicpolymers, etc.

In some embodiments, a sieving agent comprises linear or branchedpolymers. In some embodiments, a sieving agent comprises charged orneutral polymers.

In some embodiments, the sieving agent can include a blend of one ormore polymers, each having an average molecular weight and viscosity.

In some embodiments, the sieving agent comprises a polymer having anaverage molecular weight of about 10,000-2,000,000, or about12,000-95,000, or about 13,000-95,000.

In some embodiments, a sieving agent can exhibit an average viscosityrange of about 5 centipoise to about 15,000 centipoise when dissolved inwater at 2 weight percent measured at about 25° C., or about 10centipoise to about 10,000 centipoise as a 2% aqueous solutions measuredat about 25° C., or about 15 centipoise to about 5,000 centipoise as a2% aqueous solution measured at about 25° C.

In some embodiments, a sieving agent comprises a viscosity averagemolecular weight (M_(v)) of about 25 to about 1,5000 kM_(v), or about75-1,000 kM_(v), or about 85-800 kM_(v). In some embodiments, thereaction mixture comprises a sieving agent at about 0.1 to about 20%weight per volume, or about 1-10% w/v, or about 2-5% w/v.

In some embodiments, a sieving agent comprises a polysaccharide polymer.In some embodiments, a sieving agent comprises a polymer of glucose orgalactose. In some embodiments, a sieving agent comprises one or morepolymers selected from the group consisting of: cellulose, dextran,starch, glycogen, agar, chitin, pectin or agarose. In some embodiments,the sieving agent comprises a glucopyranose polymer.

In some embodiments, the sieving agent includes a cellulose derivative,such as sodium carboxy methyl cellulose, sodium carboxymethyl2-hydroxyethyl cellulose, methyl cellulose, hydroxyl ethyl cellulose,2-hydroxypropyl cellulose, carboxy methyl cellulose, hydroxyl propylcellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose,(hydroxypropyl)methyl cellulose or hydroxyethyl ethyl cellulose, or amixture including any one or more of such polymers.

In some embodiments, the reaction mixture comprises a mixture ofdifferent sieving agents, for example, a mixture of different cellulosederivatives, starch, polyacrylamide, and the like.

In some embodiments, the reaction mixture can include a crowding agent.

In some embodiments, the reaction mixture includes both a crowding agentand a sieving agent.

In some embodiments, the reaction mixture includes at least onediffusion-reducing agent. In some embodiments, a diffusion-reducingagent comprises any compound that reduces migration of polynucleotidesfrom a region of higher concentration to one having a lowerconcentration. In some embodiments, a diffusion reducing agent comprisesany compound that reduces migration of any component of a nucleic acidamplification reaction irrespective of size.

It should be noted that the concepts of a sieving agent and adiffusion-reducing agent are not necessarily mutually exclusive; asieving agent can frequently be effective in reducing diffusion oftarget compounds through a reaction mixture, whereas a diffusionreducing agent can frequently have a sieving effect on reactioncomponents. In some embodiments, the same compound or reaction mixtureadditive can act both as a sieving agent and/or a diffusion reducingagent. Any of the sieving agents disclosed herein can in someembodiments be capable of acting as a diffusion reducing agent and viceversa.

In some embodiments, the diffusion reducing agent and/or sieving agentincludes polyacrylamide, agar, agarose or a cellulose polymer such ashydroxyethyl cellulose (HEC), methyl-cellulose (MC) or carboxymethylcellulose (CMC).

In some embodiments, the sieving agent and/or the diffusion reducingagent is included in the reaction mixture at concentrations of at least1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 74%, 90%, or 95% w/v(weight of agent per unit volume of reaction mixture).

In some embodiments, the reaction mixture includes at least one crowdingagent. For example, a crowding agent can increase the concentration ofone or more components in a nucleic acid amplification reaction bygenerating a crowded reaction environment. In some embodiments, thereaction mixture includes both a sieving agent and/or diffusion reagentand a crowding agent.

In some embodiments, the nucleic acid amplification methods compriseforming a reaction mixture by combining a nucleic acid template having aforward primer binding sequence and a reverse primer binding sequence, apolymerase, a recombinase, a single-stranded binding protein, arecombinase loading protein, a blocked forward primer, a blocked reverseprimer, dNTPs, an RNase H enzyme, and a buffer comprising a divalentcation. In some embodiments, the forward primer binding sequence iscomplementary or identical to at least a portion of the blocked forwardprimer and the reverse primer binding sequence is complementary oridentical to at least a portion of the blocked reverse primer.

In some embodiments, the blocked forward primer and the blocked reverseprimer comprise a 5′ domain and a 3′ domain separated by a nucleotidecomprising a ribobase, wherein the 5′ domain is 10 to 40 nucleotides inlength and the 3′ domain is 10 to 25 nucleotides in length. In someembodiments, the blocked forward primer and the blocked reverse primercomprise a 5′ domain and a 3′ domain separated by a nucleotidecomprising a ribobase, wherein the 5′ domain is 10 to 100 nucleotides inlength and the 3′ domain is 10 to 30 nucleotides in length.

In some embodiments, the reaction mixture further comprises arecombinase accessory protein. In some embodiments, the recombinaseaccessory protein is a single-stranded binding protein and/or arecombinase loading protein.

In some embodiments, the reaction mixture comprises a blocked primerwherein 5′ domain is 15 to 30 nucleotides in length. In someembodiments, the 5′ domain of the blocked primers is 15 to 50nucleotides in length. In some embodiments, the reaction mixtureincludes a blocked primer wherein the 3′ domain is 14 to 25 nucleotidesin length. In some embodiments, the 3′ domain is 15 to 25 nucleotides inlength. In some embodiments, the 5′ domain can be at least 15nucleotides and the 3′ domain can be at least 10 nucleotides, whereinthe length of the primer does not exceed 100, 90, 80, 75, 70, 60, or 50nucleotides. In some embodiments, a 3′ nucleotide of the 3′ domain ofthe forward primer is mismatched to the forward primer binding sequence.

In some embodiments, the ribobase separating the 5′ domain and the 3′domain of the blocked primer comprises rU, rG or rA. In someembodiments, the ribobase separating the 5′ domain and the 3′ domain ofthe blocked primer comprises rC. In some embodiments, the 3′ domain ofthe blocked primers is 14 to 20 nucleotides in length and the ribobaseis rU, rG, rC or rA.

In some embodiments, the reaction mixture comprises a recombinaseaccessory protein that is uvsY. In some embodiments, the reactionmixture comprises a recombinase selected from the group consisting ofuvsX, RecA, RadA, RadB, Rad 51, a homologue thereof, a functional analogthereof and a combination thereof. In some embodiments, the reactionmixture comprises uvsY recombinase accessory protein and uvsXrecombinase.

In some embodiments, the reaction mixture comprises RNase H enzyme thatis RNase HII. In some embodiments, the RNase H enzyme is present at aconcentration from 20 U to 100 U/50 μL. In some embodiments, the RNase Henzyme is present at a concentration from 40 to 90 U/50 μL.

In some embodiments, the nucleic acid template is a member of a nucleicacid library comprising a population of nucleic acid templates eachcomprising a forward primer binding sequence, and wherein the blockedforward primer is a blocked universal forward primer that binds theuniversal forward primer binding sequence. In some embodiments, thenucleic acid templates each comprises a reverse universal primer bindingsequence and wherein the blocked reverse primer is a blocked universalreverse primer that binds the universal reverse primer binding sequence.

In some embodiments, either or both of the blocked forward primer andthe blocked reverse primer are immobilized on a solid support. In someembodiments, the solid support is a bead.

In some embodiments, the nucleic acid amplification methods compriseforming a reaction mixture by combining at least two differentpolynucleotide templates comprising both a first primer binding sequenceand a second primer binding sequence, a recombinase, a recombinaseaccessory protein, a polymerase, a first blocked universal primer, asecond blocked universal primer, dNTPs, an RNase H enzyme, and a buffer.The reaction mixture is in contact with a support having the firstblocked universal primer bound thereto, wherein the first primer bindingsequence is complementary or identical to at least a portion of thefirst blocked universal primer and the second primer binding sequence iscomplementary or identical to at least a portion of the second blockeduniversal primer.

In some embodiments, at least two substantially monoclonal nucleic acidpopulations are formed by using the polymerase to amplify each of saidat least two different polynucleotide templates onto different sites onthe solid support under substantially isothermal conditions.

In some embodiments, the second blocked universal primer is in solution(e.g., soluble primers). In some embodiments, the second blockeduniversal primer is immobilized on the support.

In some embodiments, the reaction mixture and the at least twosubstantially monoclonal nucleic acid populations are formed in the samesingle continuous liquid phase. In some embodiments, the reactionmixture and the at least two substantially monoclonal nucleic acidpopulations are formed in a water-in-oil emulsion.

In some embodiments, the at least two different polynucleotide templatesare members of a polynucleotide library, wherein each member of thepolynucleotide library comprises the first primer binding sequence andthe second primer binding sequence. In some embodiments, nucleic acidsof the at least two substantially monoclonal nucleic acid populationsare sequenced.

In some embodiments, the first blocked universal primer and the secondblocked universal primer comprise a 5′ domain and a 3′ domain separatedby a nucleotide comprising a ribobase, wherein the 5′ domain is 10 to 40nucleotides in length and the 3′ domain is 10 to 25 nucleotides inlength. In some embodiments, the reaction mixture comprises a first andsecond blocked primer wherein the 5′ domain is 15 to 30 nucleotides inlength. In some embodiments, the 5′ domain of the blocked primers is 15to 50 nucleotides in length. In some embodiments, the reaction mixturecomprises a blocked primer wherein the 3′ domain is 14 to 25 nucleotidesin length. In some embodiments, the 3′ domain is 15 to 25 nucleotides inlength. In some embodiments, a 3′ nucleotide of the 3′ domain of thefirst primer is mismatched to the first primer binding sequence.

In some embodiments, the ribobase separating the 5′ domain and the 3′domain of the blocked primer comprises rU, rG or rA. In embodiments theribobase separating the 5′ domain and the 3′ domain of the blockedprimer comprises rC. In some embodiments, the 3′ domain of the blockedprimers is 14 to 20 nucleotides in length and the ribobase is rU, rG, rCor rA.

In some embodiments, the reaction mixture comprises a recombinaseaccessory protein that is uvsY. In some embodiments, the reactionmixture comprises a recombinase selected from the group consisting ofuvsX, RecA, RadA, RadB, Rad 51, a homologue thereof, a functional analogthereof and a combination thereof. In some embodiments, the reactionmixture comprises uvsY recombinase accessory protein and uvsXrecombinase.

In some embodiments, the reaction mixture comprises RNase H enzyme thatis RNase HII. In some embodiments, the RNase H enzyme is present at aconcentration from 20 U to 100 U/50 μL. In some embodiments, the RNase Henzyme is present at a concentration from 40 to 90 U/50 μL.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits, wherein the nucleic acidamplification includes the amplification reaction mixture which caninclude one or more solid or semi-solid supports. In some embodiments,at least one of the supports can include one or more instances of afirst blocked primer including a first primer sequence. As used herein,the blocked primers refer to those described above containing a 5′domain, at least one ribobase, a 3′ domain and a blocking group. Theappropriate length for the 5′ and 3′ domain the primers are described indetail above, for example the 3′ domain has a length of at least 10nucleotides such as 10 to 30 nucleotides. In the section that follows,the primers that may be attached to a support or surface are thoseblocked primers described herein. For example, a forward or reverseblocked primer is attached to a solid support via the 5′ end of theprimer. See FIG. 11 for exemplary blocked primers for use on a solidsupport.

In some embodiments, at least one polynucleotide template in thereaction mixture includes a first primer binding sequence. The firstprimer binding sequence can be substantially identical or substantiallycomplementary to the first blocked primer sequence. In some embodiments,at least one, some or all of the supports include a plurality of firstblocked primers that are substantially identical to each other. In someembodiments, all of the blocked primers on the supports aresubstantially identical to each other, or all include a substantiallyidentical first primer sequence.

In some embodiments, at least one of the supports includes two or moredifferent blocked primers attached thereto. For example, the at leastone support can include at least one instance of the first blockedprimer and at least one instance of a second blocked primer.

In some embodiments, the aqueous phase of the reaction mixture includesa plurality of supports, at least two supports of the plurality beingattached to blocked primers including a first priming sequence. In someembodiments, the reaction mixture includes two or more differentpolynucleotide templates having a first primer binding sequence.

Alternatively, in some embodiments, the reaction mixture does notinclude any supports. In some embodiments, the at least two differentpolynucleotide templates are amplified directly onto a surface of thesite or reaction chamber of the array. In some embodiments, the reactionchambers are arranged in an array on a support and the reaction chambersare used to conduct sequencing reactions. In some embodiments, thereaction chambers are arranged in an array on a sequencing support.

In some embodiments, methods for nucleic acid amplification comprise oneor more surfaces. In some embodiments, a surface can be attached with aplurality of first primers, the first primers of the plurality sharing acommon first primer sequence.

In some embodiments, a surface can be an outer or top-most layer orboundary of an object. In some embodiments, a surface can be interior tothe boundary of an object.

In some embodiments, the reaction mixture includes multiple differentsurfaces, for example, the reaction mixture can include a plurality ofbeads (such as particles, nanoparticles, microparticles, and the like)and at least two different polynucleotide templates can be clonallyamplified onto different surfaces, thereby forming at least twodifferent surfaces, each of which is attached to an amplicon. In someembodiments, the reaction mixture includes a signal surface (forexample, the surface of a slide or array of reaction chambers) and atleast two different polynucleotide templates are amplified onto twodifferent regions or locations on the surface, thereby forming a singlesurface attached to two or more amplicons.

In some embodiments, a surface can be porous, semi-porous or non-porous.In some embodiments, a surface can be a planar surface, as well asconcave, convex, or any combination thereof. In some embodiments, asurface can be a bead, particle, microparticle, sphere, filter,flowcell, well, groove, channel reservoir, gel or inner wall of acapillary. In some embodiments, a surface includes the inner walls of acapillary, a channel, a well, groove, channel, reservoir. In someembodiments, a surface can include texture (e.g., etched, cavitated,pores, three-dimensional scaffolds or bumps).

In some embodiments, a surface can be magnetic or paramagnetic bead(e.g., magnetic or paramagnetic nanoparticles or microparticles). Insome embodiments, paramagnetic microparticles can be paramagnetic beadsattached with streptavidin (e.g., Dynabeads™ M-270 from Invitrogen,Carlsbad, Calif.). Particles can have an iron core, or comprise ahydrogel or agarose (e.g., Sepharose™).

In some embodiments, the surface can have immobilized thereon, aplurality of an RNase-cleavable first blocked primer. See Example 5. Asurface can be coated with an acrylamide, carboxylic or amine compoundfor attaching a nucleic acid (e.g., a first primer). In someembodiments, an amino-modified nucleic acid (e.g., primer) can beattached to a surface that is coated with a carboxylic acid. In someembodiments, an amino-modified nucleic acid can be reacted with EDC (orEDAC) for attachment to a carboxylic acid coated surface (with orwithout NHS). A first blocked primer can be immobilized to an acrylamidecompound coating on a surface. Particles can be coated with anavidin-like compound (e.g., streptavidin) for binding biotinylatednucleic acids.

In some embodiments, the surface comprises the surface of a bead. Insome embodiments, a bead comprises a polymer material. For example, abead comprises a gel, hydrogel or acrylamide polymers. A bead can beporous. Particles can have cavitation or pores, or can includethree-dimensional scaffolds. In some embodiments, particles can be IonSphere™ particles.

In some embodiments, the disclosed methods (as well as relatedcompositions, systems and kits) include immobilizing one or more nucleicacid templates onto one or more supports. Nucleic acids may beimmobilized on the solid support by any method including but not limitedto physical adsorption, by ionic or covalent bond formation, orcombinations thereof. A solid support may include a polymeric, a glass,or a metallic material. Examples of solid supports include a membrane, aplanar surface, a microtiter plate, a bead, a filter, a test strip, aslide, a cover slip, and a test tube. A solid support means any solidphase material upon which an oligomer is synthesized, attached, ligatedor otherwise immobilized. A support can optionally comprise a “resin”,“phase”, “surface” and “support”. A support may be composed of organicpolymers such as polystyrene, polyethylene, polypropylene,polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well asco-polymers and grafts thereof. A support may also be inorganic, such asglass, silica, controlled-pore-glass (CPG), or reverse-phase silica. Theconfiguration of a support may be in the form of beads, spheres,particles, granules, a gel, or a surface. Surfaces may be planar,substantially planar, or non-planar. Supports may be porous ornon-porous, and may have swelling or non-swelling characteristics. Asupport can be shaped to comprise one or more wells, depressions orother containers, vessels, features or locations. A plurality ofsupports may be configured in an array at various locations. A supportis optionally addressable (e.g., for robotic delivery of reagents), orby detection means including scanning by laser illumination and confocalor deflective light gathering. An amplification support (e.g., a bead)can be placed within or on another support (e.g., within a well of asecond support).

In some embodiments, the solid support is a “microparticle,” “bead,”“microbead,” etc., (optionally but not necessarily spherical in shape)having a smallest cross-sectional length (e.g., diameter) of 50 micronsor less, preferably 10 microns or less, 3 microns or less, approximately1 micron or less, approximately 0.5 microns or less, e.g., approximately0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer,about 1-10 nanometer, about 10-100 nanometers, or about 100-500nanometers). Microparticles (e.g., Dynabeads from Dynal, Oslo, Norway)may be made of a variety of inorganic or organic materials including,but not limited to, glass (e.g., controlled pore glass), silica,zirconia, cross-linked polystyrene, polyacrylate, polymehtymethacrylate,titanium dioxide, latex, polystyrene, etc. Magnetization can facilitatecollection and concentration of the microparticle-attached reagents(e.g., polynucleotides or ligases) after amplification, and can alsofacilitate additional steps (e.g., washes, reagent removal, etc.). Insome embodiments of the invention a population of microparticles havingdifferent shapes sizes and/or colors can be used. The microparticles canoptionally be encoded, e.g., with quantum dots such that eachmicroparticle can be individually or uniquely identified.

In some embodiments, a bead surface can be functionalized for attachinga plurality of a first blocked primer. In some embodiments, a bead canbe any size that can fit into a reaction chamber. For example, one beadcan fit in a reaction chamber. In some embodiments more than one beadcan fit in a reaction chamber. In some embodiments, the smallestcross-sectional length of a bead (e.g., diameter) can be about 50microns or less, or about 10 microns or less, or about 3 microns orless, approximately 1 micron or less, approximately 0.5 microns or less,e.g., approximately 0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g.,under 1 nanometer, about 1-10 nanometer, about 10-100 nanometers, orabout 100-500 nanometers). In some embodiments, a bead can be attachedwith a plurality of one or more different blocked primer sequences. Insome embodiments, a bead can be attached with a plurality of one blockedprimer sequence, or can be attached a plurality of two or more differentblocked primer sequences. In some embodiments, a bead can be attachedwith a plurality of at least 1,000 primers, or about 1,000-10,000primers, or about, 10,000-50,000 primers, or about 50,000-75,000primers, or about 75,000-100,000 primers, or more. In some embodiments,the disclosure relates generally to methods, as well as relatedcompositions and kits, wherein the nucleic acid amplification includesthe reaction mixtures discussed in the context of methods providedherein themselves form embodiments of the invention. In someembodiments, the compositions that include a recombinase, a polymerasesuitable for RPA, and an RNase H enzyme that is active at temperaturesat which the recombinase and polymerase are active, and is compatiblewith those enzymes. In some embodiments, the compositions can furtherinclude a single stranded binding protein and/or a recombinase loadingprotein. The recombinase and polymerase are typically present ateffective concentrations for recombinase polymerase amplification, or athigher concentrations such that they can be combined with other reactioncomponents into a final blocked primer-RPA reaction mixture. RNase ispresent at an effective concentration, such as a limiting and/especiallyan excess concentration, as disclosed herein, or 2×, 3×, 4×, 5×, or 10×such concentrations. The RNase can be any of the RNases discussedherein, including in illustrative examples, E. coli RNase HII.

In some embodiments, the compositions can further include othercomponents of an RPA and/or RNase reaction. For example, thecompositions can include dNTPs and a buffer. In addition, thecomposition can include a blocked forward primer and a blocked reverseprimer. As a non-limiting example, the composition can include arecombinase, a polymerase, an RNase H enzyme that is other than athermostable RNase H, a nucleic acid template, uvsX recombinase, uvsYrecombinase loading protein, gp32 protein, Sau DNA polymerase, dNTPs,ATP, phosphocreatine and creatine kinase. In some embodiments, thecomposition can be in liquid form, or it can be in a solid form, such asa dried-down pellet form that can be rehydrated. Furthermore, componentsfor compositions provided herein, can be split up such that anycombination of the components can be in a pellet or liquid form, and oneor more combinations of the rest of the components can be in one or moreseparate pellet or liquid forms. Such combinations can form kits thatinclude at least two of such combinations. For example, a kit of theinvention can include a pellet that includes all the reaction mixturecomponents provided herein except for the RNase enzyme, which can beprovided in a separate pellet or liquid in the kit.

In some embodiments, provided herein are compositions and kits includingat least one blocked primer of the invention that includes aribonucleotide as disclosed in detail herein. The compositions and kitscan further include a second blocked primer or a standard primer. Insome embodiments, the compositions and kits include a pair of primers(forward and reverse) wherein at least one is a blocked primer of theinvention.

In some embodiments, the composition includes a reaction mixture havingat least a blocked primer that includes a ribonucleotide as discussed indetail herein, and a recombinase. In some embodiments, the compositionfurther includes amplification reagents including template nucleic acid,polymerase, RNase H, and/or accessory proteins. The reaction mixture foran amplification reaction typically includes a source of nucleotides, oranalogs thereof, that is used by the polymerase as substrates for anextension reaction.

In some embodiments, a composition comprises nucleic acid template, apolymerase, a recombinase, a blocked forward primer, a blocked reverseprimer, dNTPs, an RNase H enzyme, and a buffer. In some embodiments, thecomposition comprises a nucleic acid template, blocked forward primer, ablocked reverse primer, uvsX recombinase, uvsY recombinase loadingprotein, gp32 protein, Sau DNA polymerase, dNTPs, ATP, phosphocreatineand creatine kinase.

In some embodiments, the blocked forward primer and/or the blockedreverse primer in compositions, reaction mixtures, and kits of theinvention can include any of the blocked primers disclosed herein. Forexample, the blocked forward primer and/or the blocked reverse primercan include a 5′ domain and a 3′ domain separated by a nucleotidecomprising a ribobase, wherein the 5′ domain is 10 to 40 nucleotides inlength and the 3′ domain is 11 to 25 nucleotides in length. In someembodiments, the composition comprises a forward and reverse blockedprimer wherein the 5′ domain is 15 to 30 nucleotides in length. In someembodiments, the 5′ domain of the blocked primers is 15 to 50nucleotides in length. In some embodiments, the composition comprises ablocked primer wherein the 3′ domain is 14 to 25 nucleotides in length.In some embodiments, the 3′ domain is 15 to 25 nucleotides in length. Insome embodiments, a 3′ nucleotide of the 3′ domain of the forward primeris mismatched to the forward primer binding sequence.

In some embodiments, a composition of the invention comprises at leasttwo different polynucleotide templates comprising both a first primerbinding sequence and a second primer binding sequence, a recombinase, arecombinase accessory protein, a polymerase, a first blocked universalprimer, a second blocked universal primer, dNTPs, an RNase H enzyme, anda buffer. In some embodiments, the composition further comprises asupport. In some embodiments, the support is a bead. In furtherembodiments, the first blocked universal primer is attached to the beadsupport.

In some embodiments, the composition includes at least two differentpolynucleotide templates comprising both a first primer binding sequenceand a second primer binding sequence, uvsX recombinase, uvsY recombinaseloading protein, gp32 protein, Sau DNA polymerase, APT, phosphocreatine,creatine kinase, a first blocked universal primer attached to a beadsupport, a second blocked universal primer, an RNase H enzyme, and abuffer.

In some embodiments, the first blocked universal primer and the secondblocked universal primer comprise a 5′ domain and a 3′ domain separatedby a nucleotide comprising a ribobase, wherein the 5′ domain is 10 to 40nucleotides in length and the 3′ domain is 10 to 25 nucleotides inlength. In some embodiments, the composition comprises a first andsecond blocked primer wherein the 5′ domain is 15 to 30 nucleotides inlength. In some embodiments, the 5′ domain of the blocked primers is 15to 50 nucleotides in length. In some embodiments, the compositioncomprises a blocked primer wherein the 3′ domain is 14 to 25 nucleotidesin length. In some embodiments, the 3′ domain is 15 to 25 nucleotides inlength. In some embodiments, a 3′ nucleotide of the 3′ domain of thefirst primer is mismatched to the first primer binding sequence.

In some embodiments, compositions are also amendable to kit formatwherein the primers, and amplification may be in the same contain,separate contains and in liquid or dehydrated form. The kit may compriseinstructions for performing the RPA methods for amplification of nucleicacid template including clonal amplification for downstream sequencingmethods. In one embodiment, the kit provides instructions for nucleicacid sequencing preparation.

In some embodiments, provided herein is a kit that includes at least twocontainers at least one of which includes a blocked, at least one ofwhich includes a recombinase and at least one of which includes an RNaseH. The recombinase, RNase H and the blocked can be in the same ordifferent tubes.

In some embodiments, at least one blocked primer can be attached to asupport. In some embodiments, the kit comprises at least one blockedprimer attached to a bead support.

In some embodiments, the container comprising the recombinase furthercomprises one or more amplification reagents including a recombinaseaccessory protein, a polymerase, dNTPs, an RNase H enzyme, and a buffer.In some embodiments the kit comprises one or more containers comprisinguvsX recombinase, uvsY recombinase loading protein, gp32 protein, SauDNA polymerase, dNTPs, RNase H, ATP, phosphocreatine and creatinekinase.

In some embodiments, the kit comprises a blocked forward primer and ablocked reverse primer comprising a 5′ domain and a 3′ domain separatedby a nucleotide comprising a ribobase, wherein the 5′ domain is 10 to 40nucleotides in length and the 3′ domain is 10 to 25 nucleotides inlength. In some embodiments, the kit comprises a forward and reverseblocked primer wherein the 5′ domain is 15 to 30 nucleotides in length.In some embodiments, the 5′ domain of the blocked primers is 15 to 50nucleotides in length. In some embodiments, the kit comprises a blockedprimer wherein the 3′ domain is 14 to 25 nucleotides in length. In someembodiments, the 3′ domain is 15 to 25 nucleotides in length. In someembodiments, a 3′ nucleotide of the 3′ domain of the forward primer ismismatched to the forward primer binding sequence.

In some embodiments, the kit the first blocked universal primer and thesecond blocked universal primer comprise a 5′ domain and a 3′ domainseparated by a nucleotide comprising a ribobase, wherein the 5′ domainis 10 to 40 nucleotides in length and the 3′ domain is 10 to 25nucleotides in length. In some embodiments, the composition comprises afirst and second blocked primer wherein the 5′ domain is 15 to 30nucleotides in length. In some embodiments, the 5′ domain of the blockedprimers is 15 to 50 nucleotides in length. In some embodiments, the kitcomprises a blocked primer wherein the 3′ domain is 14 to 25 nucleotidesin length. In some embodiments, the 3′ domain is 15 to 25 nucleotides inlength. In some embodiments, a 3′ nucleotide of the 3′ domain of thefirst primer is mismatched to the first primer binding sequence.

In some embodiments, the kit comprises a first blocked universal primerattached to a bead support. In some embodiments, the kit furthercomprises instructions for clonal amplification of two or nucleic acidtemplates to be used for nucleic acid sequencing.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, kits, systems and apparatuses, for nucleicacid amplification, comprising amplifying a nucleic acid template toproduce an amplicon using the RNase-cleavable blocked primers disclosedherein. In some embodiments, the amplicon is a substantially monoclonalpopulation of polynucleotides. Monoclonality can be desirable in nucleicacid assays because the different characteristics of the diversepolynucleotides within a polyclonal population can complicate theinterpretation of assay data. One example involves nucleic acidsequencing applications, in which the presence of polyclonal populationscan complicate the interpretation of sequencing data; however, with manysequencing systems are not sensitive enough to detect nucleotidesequence data from a single polynucleotide template, thus requiringclonal amplification of templates prior to sequencing.

In some embodiments, the amplification methods can be employed toclonally amplify two or more different nucleic acid templates,optionally using and within the same reaction mixture, to produce atleast two substantially monoclonal, and in some embodiments, monoclonalnucleic acid populations. Optionally, at least one of the substantiallymonoclonal populations is formed via amplification of a singlepolynucleotide template.

In some embodiments, the reaction mixture can be incubated undersubstantially isothermal amplification conditions thereby amplifying thenucleic acid template(s). In some embodiments, the isothermal conditionsare typically between 20° C. to 50° C., in some embodiments 20° C. to45° C., in some embodiments, 20° C. to 45° C., in other embodiments, 25°C. to 40° C., and still other embodiments 25° C. to 37° C. for 2 to 240minutes. In some embodiments, the temperature is between 30° C. and 42°C. In some embodiments, the reaction mixture is not exposed to atemperature above 40° C., or above 41° C., or above 42° C., or above 43°C., or above 45° C. or not exposed to a temperature above 50° C. In someembodiments, the reaction mixture is not exposed to hot startconditions. A rate limiting enzyme may be RNase H, wherein a highconcentration or excess (i.e. non-limiting) amount of the endonucleaseensures the amplification reaction proceeds based on the kinetics of thepolymerase. See Example 3.

As illustrated in FIG. 1, once blocked primers hybridize tocomplementary template sequences, RNase H enzyme is activated, cleavingthe ribonucleotide linkage in the blocked primer present in duplex DNA.The 3′ domain comprising the blocking group dissociates, liberating theblocking group which blocks amplification, creating a free 3′-hydroxylwhich is now capable of primer extension. Alternatively, RNase nicks theDNA, and the 3′ domain comprising the blocking group is displaced by the5′ domain primer extension. RNase H enzyme used here is active atbetween about 20° C. to 45° C., the temperature range for the RPAamplification methods. One drawback to the use of recombinaseamplification methods is that primer/primer hybrids are typically stableat these temperatures, leading to primer artifact amplification.However, the use of the blocked primers comprising a ribonucleasecleavage location reduces or eliminates primer dimer productamplification. See Example 2.

In some embodiments, the amplification is typically performed undersubstantially isothermal amplification conditions. The substantiallyisothermal temperature can be between 20, 21, 22, 23, 24, 25, 30, 35 or40 on the low end of the range, and 21, 22, 23, 24, 25, 26, 30, 35, 40or 45 on the high end of the range. In some embodiments, the temperatureis between 20° C. and 45° C. In some embodiments, the temperature isbetween 35° C. and 45° C. In some embodiments, the temperature is 37° C.

In some embodiments, an isothermal RPA nucleic acid amplificationreaction can be conducted at about 15-25° C., or about 25-35° C., orabout 35-40° C., or about 35-42° C., or about 40-45° C., or about 45-50°C., or about 50-55° C., or about 55-60° C. However, it is understood theenzymes used at these temperatures will need to be optimized incombination and may require changes in the enzyme, for example the DNApolymerase used, Bst instead of Bsu, or the RNase H enzyme, such asRNase H2 instead of RNase HII.

In some embodiments, any of the nucleic acid amplification methodsdisclosed herein can be conducted, or can include steps that areconducted, under isothermal or substantially isothermal amplificationconditions. In some embodiments isothermal amplification conditionscomprise a nucleic acid amplification reaction subjected to atemperature variation which is constrained within a limited range duringat least some portion of the amplification (or the entire amplificationprocess), including for example a temperature variation is equal or lessthan about 20° C., or about 10° C., or about 5° C., or about 1-5° C., orabout 0.1-1° C., or less than about 0.1° C., or, for example atemperature variation is equal or less than 20° C., or 10° C., or 5° C.,or 1-5° C., or 0.1-1° C., or less than 0.1° C.

The amplification can be carried out for 2 minutes to 240 minutes,thereby amplifying the nucleic acid template. In some embodiments, thereaction time is between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 25, 40, 50, 60, 70, 80, 90, 100, 120, 140,160, 180, 200 or 220 minutes on the low end of the range, and 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28,29, 30, 25, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220 or240 minutes on the high end of the range. In some embodiments, thereaction mixture is incubated to generate an amplified template for atleast 5 minutes, wherein reaction time is between 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200 or 220 minutes on the low end of the range,and 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27,28, 29, 30, 25, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,220 or 240 minutes on the high end of the range.

In some embodiments, the reaction mixture is incubated from 5 to 60minutes. In some embodiments, the amplifying time is from 15 to 60minutes. In some embodiments, the amplifying time is from 15 to 45minutes. In some embodiments, the reaction mixture is incubated for 30minutes to generate an amplified template sequence. In some embodiments,the reaction mixture is incubated for 50 minutes to generate anamplified template sequence.

In some embodiments, an isothermal nucleic acid amplification reactioncan be conducted for about 2, 5, 10, 15, 20, 30, 40, 50, 60 or 120minutes.

In some embodiments, the reaction mixture is formed by combining anucleic acid template having a forward primer binding sequence and areverse primer binding sequence, with the following optional reagents: apolymerase, a recombinase, a single-stranded binding protein, arecombinase loading protein, a blocked forward primer, a blocked reverseprimer, dNTPs, ATP, phosphocreatine and creatine kinase, an RNase Henzyme, and a buffer, and wherein a divalent cation, such as MgCl₂ orMg(OAc)₂ can be added to start the reaction. In some embodiments, thebuffer may include a crowding agent, such as PEG, Tris buffer and apotassium acetate salt. The forward primer binding sequence iscomplementary or identical to at least a portion of the blocked forwardprimer and the reverse primer binding sequence is complementary oridentical to at least a portion of the blocked reverse primer. Thereaction mixture is incubated under substantially isothermalamplification conditions, for example between 35° C. and 45° C. for 15to 60 minutes, thereby amplifying the nucleic acid template.

In some embodiments, the blocked forward primer and the blocked reverseprimer comprise a 5′ domain and a 3′ domain separated by a nucleotidecomprising a ribobase, wherein the 5′ domain is 10 to 40 nucleotides inlength and the 3′ domain is 11 to 25 nucleotides in length. The forwardand reverse primers, in some embodiments, bind in opposite directions todifferent strands of a double-stranded template, such that the regionbetween the primer binding sites of the template is amplified, as isknown for pairs of amplification primers.

In some embodiments, amplification methods and associated compositionsprovided herein that include improved ribobase-containing primers, canbe used in a nucleic acid sequencing workflow, especially a highthroughput nucleic acid sequencing workflow. In some embodiments, thereaction mixture is formed by combining at least two differentpolynucleotide templates comprising both a first primer binding sequenceand a second primer binding sequence, a recombinase, a recombinaseaccessory protein, a polymerase, a first blocked universal primer, asecond optionally blocked universal primer, dNTPs, an RNase H enzyme,and a buffer. In some embodiments, the reaction mixture is in contactwith a support having the first blocked universal primer bound thereto,wherein the first primer binding sequence is complementary or identicalto at least a portion of the first blocked universal primer and thesecond primer binding sequence is complementary or identical to at leasta portion of the second optionally blocked universal primer.

The nucleic acid amplification methods result in the formation of atleast two substantially monoclonal nucleic acid populations by using thepolymerase to amplify each of the at least two different polynucleotidetemplates onto different sites on the solid support, within the samereaction mixture of step (a) under substantially isothermal conditions.

In some embodiments, the two or more different nucleic acid templatesare amplified simultaneously and/or in parallel.

In some embodiments, the second optionally blocked universal primer isin solution (e.g., soluble primers). In some embodiments, the secondoptionally blocked universal primer is immobilized on the support.

In some embodiments, the reaction mixture and the at least twosubstantially monoclonal nucleic acid populations are formed in the samesingle continuous liquid phase. In some embodiments, the reactionmixture and the at least two substantially monoclonal nucleic acidpopulations are formed in a water-in-oil emulsion.

In some embodiments, the at least two different polynucleotide templatesare members of a polynucleotide library, wherein each member of thepolynucleotide library comprises the first primer binding sequence andthe second primer binding sequence. In some embodiments, nucleic acidsof the at least two substantially monoclonal nucleic acid populationsare sequenced.

In some embodiments, the first blocked universal primer and the secondoptionally blocked universal primer when it is present in a blockedconfiguration comprise a 5′ domain and a 3′ domain separated by anucleotide comprising a ribobase, wherein the 5′ domain is 10 to 40nucleotides in length and the 3′ domain is 10 to 25 nucleotides inlength. In some embodiments, embodiments, the reaction mixture comprisesa first and second blocked primer wherein the 5′ domain is 15 to 30nucleotides in length. In some embodiments, the 5′ domain of the blockedprimers is 15 to 50 nucleotides in length. In some embodiments, thereaction mixture comprises a blocked primer wherein the 3′ domain is 14to 25 nucleotides in length. In some embodiments, the 3′ domain is 15 to25 nucleotides in length. In some embodiments, a 3′ nucleotide of the 3′domain of the forward primer is mismatched to the forward primer bindingsequence.

In some embodiments, the ribobase separating the 5′ domain and the 3′domain of the blocked primer comprises rU, rG or rA. In someembodiments, the ribobase separating the 5′ domain and the 3′ domain ofthe blocked primer comprises rC. In some embodiments, the 3′ domain ofthe blocked primers is 14 to 20 nucleotides in length and the ribobaseis rU, rG, rC or rA.

In some embodiments, the reaction mixture used in the methods providedherein is a composition section provided herein. The reaction mixturecan include components such as, for example, a recombinase accessoryprotein such as uvsY at concentrations provided herein. For example, theuvsY can be present, at 20 ng/ul to 100 ng/ul. In some embodiments, thereaction mixture comprises a recombinase selected from the groupconsisting of uvsX, RecA, RadA, RadB, Rad 51, a homologue thereof, afunctional analog thereof and a combination thereof. The UvsX proteincan be present, for example, at 50-250 ng/ul or 100-200 ng/ul. In someembodiments, the reaction mixture comprises uvsY recombinase accessoryprotein and uvsX recombinase.

In some embodiments, the reaction mixture comprises an RNase H enzymeaccording to any of the teachings provided in the RNase section herein.For example, the RNase H enzyme can be E. coli RNase HII. In someembodiments, the RNase H enzyme is present at a limiting or especiallyan excess concentration as provided herein. Useful concentrations forsuch RNase H enzyme is provided elsewhere herein.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems and kits) for nucleic acidsynthesis, comprising: providing at least two double stranded nucleicacid templates in a reaction mixture; and forming at least twosubstantially monoclonal nucleic acid populations by clonally amplifyingthe at least two double stranded nucleic acid templates according to anyof the methods described herein.

In some embodiments, clonally amplifying optionally includes forming areaction mixture. The reaction mixture can include a continuous liquidphase. In some embodiments, the continuous liquid phase includes asingle continuous aqueous phase. The liquid phase can include two ormore polynucleotide templates, which can optionally have the samenucleotide sequence, or can have nucleotide sequences that are differentfrom each other. In some embodiments, at least one of the two or morepolynucleotide templates can include at least one nucleic acid sequencethat is substantially non-identical, or substantially non-complementary,to at least one other polynucleotide template within the reactionmixture.

In some embodiments, the two or more different nucleic acid templatesare localized, deposited or positioned at different sites prior to theamplifying.

In some embodiments, the two or more different nucleic acid templatesare clonally amplified in solution, optionally within a single reactionmixture, and the resulting two or more substantially monoclonal nucleicacid populations are then localized, deposited or positioned atdifferent sites following such clonal amplification.

The different sites are optionally members of an array of sites. Thearray can include a two-dimensional array of sites on a surface (e.g.,of a flowcell, electronic device, transistor chip, reaction chamber,channel, and the like), or a three-dimensional array of sites within amatrix or other medium (e.g., solid, semi-solid, liquid, fluid, and thelike).

In some embodiments, the two or more different nucleic acid templatesare amplified within a continuous liquid phase, typically a continuousaqueous phase, of the same reaction mixture, thereby producing two ormore different and substantially monoclonal populations ofpolynucleotides, each population being generated via amplification of asingle polynucleotide template present in the reaction mixture.

In some embodiments, the continuous liquid phase is contained within asingle or same phase of the reaction mixture.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems and kits) for nucleic acidsynthesis, comprising: providing a double stranded nucleic acidtemplate; and forming a substantially monoclonal nucleic acid populationby amplifying the double stranded nucleic acid template. Optionally, theamplifying includes clonally amplifying the double stranded nucleic acidtemplate.

In some embodiments, the amplifying includes performing at least oneround of amplification under substantially isothermal conditions.

In some embodiments, the amplifying includes performing at least twoconsecutive cycles of nucleic acid synthesis under substantiallyisothermal conditions.

In some embodiments, the RPA methods can be used for template walking.For example, the amplifying can include performing at least one round oftemplate-walking.

In some embodiments, the amplifying optionally includes performing twodifferent rounds of amplification within the sites or reaction chambers.For example, the amplifying can include performing at least one round ofthe RPA methods within the sites or reaction chambers, and performing atleast one round of template walking, which may or may not use the RPAmethods with blocked primes, within the sites or reaction chambers, inany order or combination of rounds. In some embodiments, at least twoconsecutive cycles in any one or more of the rounds of amplification areperformed under substantially isothermal conditions. In someembodiments, at least one of the rounds of amplification is performedunder substantially isothermal conditions.

In some embodiments, the nucleic acid template to be amplified is doublestranded, or is rendered at least partially double stranded usingappropriate procedures prior to amplification. (The template to beamplified is referred to interchangeably herein as a nucleic acidtemplate or a polynucleotide template). In some embodiments, thetemplate is linear. Alternatively, the template can be circular, orinclude a combination of linear and circular regions.

In some embodiments, the double stranded nucleic acid template includesa forward strand. The double stranded nucleic acid template can furtherinclude a reverse strand. The forward strand optionally includes a firstprimer binding site. The reverse strand optionally includes a secondprimer binding site.

In some embodiments, the template already includes a first and/or secondprimer binding site. Alternatively, the template optionally does notoriginally include a primer binding site, and the disclosed methodsoptionally include attaching or introducing a primer binding site to thetemplate prior to the amplifying. For example, the method can optionallyinclude ligating or otherwise introducing an adapter containing a primerbinding site to, or into, the templates. The adapter can be ligated orotherwise introduced to an end of a linear template, or within the bodyof a linear or circular template. Optionally, the template can becircularized after the adapter is ligated or introduced. In someembodiments, a first adapter can be ligated or introduced at a first endof a linear template, and a second adaptor can be ligated or introducedat a second end of the template.

In some embodiments, the amplifying includes contacting the partiallydenatured template with a first blocked primer, with a second blockedprimer, or with both a first blocked primer and a second blocked primer,in any order or combination.

In some embodiments, the first blocked primer contains a first primersequence. The first blocked primer optionally includes an extendible end(e.g., a 3′OH containing end), after cleavage by the ribo-endonucleaseto liberate the blocking group and the 3′ domain of the blocked primers.The first blocked primer can optionally be attached to a compound (e.g.,a “drag tag”), or to a support (e.g., a bead or a surface of the site orreaction chamber).

In some embodiments, the second blocked primer contains a second primersequence. The second primer optionally includes an extendible end (e.g.,a 3′OH containing end) after cleavage by the ribo-endonuclease toliberate the blocking group and the 3′ domain of the blocked primers.The second blocked primer can optionally be attached to a compound(e.g., a “drag tag”), or to a support (e.g., a bead or a surface of thesite or reaction chamber).

Optionally, the first blocked primer binds to the first primer bindingsite to form a first primer-template duplex. The second blocked primercan bind to the second primer binding site to form a secondprimer-template duplex.

In some embodiments, amplifying includes extending the first blockedprimer (after cleavage by the ribo-endonuclease to liberate the blockinggroup and the 3′ domain of the blocked primers) to form an extendedfirst primer. For example, amplifying can include extending the firstblocked primer of the first primer-template duplex to form an extendedfirst primer.

In some embodiments, amplifying includes extending the first blockedprimer (after cleavage by the ribo-endonuclease to liberate the blockinggroup and the 3′ domain of the blocked primers) to form an extendedfirst primer. For example, amplifying can include extending the firstblocked primer of the first primer-template duplex to form an extendedfirst primer.

In some embodiments, the amplifying includes forming a partiallydenatured template. For example, the amplification can include partiallydenaturing the double stranded nucleic acid template.

In some embodiments, partially denaturing includes subjecting the doublestranded nucleic acid template to partially denaturing conditions.

In some embodiments, partially denaturing conditions include treating orcontacting the nucleic acid templates to be amplified with one or moreenzymes that are capable of partially denaturing the nucleic acidtemplate, optionally in a sequence-specific or sequence-directed manner.

In some embodiments, at least one enzyme catalyzes strand invasionand/or unwinding, optionally in a sequence-specific manner. Optionally,the one or more enzymes include one or more enzymes selected from thegroup consisting of: recombinases, topoisomerases and helicases. In someembodiments, partially denaturing the template can include contactingthe template with a recombinase and forming a nucleoprotein complexincluding the recombinase. Optionally, the template is contacted with arecombinase in the presence of a first blocked primer, a second blockedprimer, or both a first and second blocked primer. Partially denaturingcan include catalyzing strand exchange using the recombinase andhybridizing the first blocked primer to the first primer binding site(or hybridizing the second blocked primer to the second primer bindingsite). In some embodiments, partially denaturing includes performingstrand exchange and hybridizing both the first blocked primer to thefirst primer binding site and the second blocked primer to the secondprimer binding site using the recombinase.

In some embodiments, the partially denatured template includes a singlestranded portion and a double stranded portion. In some embodiments, thesingle stranded portion includes the first primer binding site. In someembodiments, the single stranded portion includes the second primerbinding site. In some embodiments, the single stranded portion includesboth the first primer binding site and the second primer binding site.

In some embodiments, partially denaturing the template includescontacting the template with one or more nucleoprotein complexes. Atleast one of the nucleoprotein complexes can include a recombinase. Atleast one of the nucleoprotein complexes can include a blocked primer(e.g., a first primer or a second primer, or a primer including asequence complementary to a corresponding primer binding sequence in thetemplate). In some embodiments, partially denaturing the template caninclude contacting the template with a nucleoprotein complex including aprimer. Partially denaturing can include hybridizing the blocked primerof the nucleoprotein complex to the corresponding primer binding site inthe template, thereby forming a primer-template duplex.

In some embodiments, partially denaturing the template can includecontacting the template with a first nucleoprotein complex including afirst blocked primer. Partially denaturing can include hybridizing thefirst blocked primer of the first nucleoprotein complex to the firstprimer binding site of the forward strand, thereby forming a firstblocked primer-template duplex.

In some embodiments, partially denaturing the template can includecontacting the template with a second nucleoprotein complex including asecond blocked primer. Partially denaturing can include hybridizing thesecond blocked primer of the second nucleoprotein complex to the secondprimer binding site of the reverse strand, thereby forming a secondprimer-template duplex.

In some embodiments, the disclosed methods (and related compositions,systems and kits) can further include one or more primer extensionsteps. For example, the methods can include extending a primer vianucleotide incorporation using a polymerase. As understood with thecurrent RPA methods and blocked primers, before primer extension canproceed the RNase H enzyme (e.g. RNase HII) cleaves the primer at theribobase location. The 5′ domain remains hybridized to the template witha 3′OH group available for primer extension, while the 3′ domaincontaining the blocking group is dispersed into the reaction mixture anddoes not participate in nucleic acid amplification.

In some embodiments, extending a primer includes contacting thehybridized primer with a polymerase and one or more types of nucleotidesunder nucleotide incorporation conditions. Typically, extending a primeroccurs in a template-dependent fashion.

In some embodiments, the methods (and related compositions, systems andkits) include extending the first primer by incorporating one or morenucleotides into the first primer of the first primer-template duplexusing the polymerase, thereby forming an extended first primer.

In some embodiments, the methods (and related compositions, systems andkits) include binding a second blocked primer to the second primerbinding site of the first extended primer by any suitable method (e.g.,ligation or hybridization).

In some embodiments, the methods (and related compositions, systems andkits) include extending the second primer by incorporating one or morenucleotides into the second primer of the second primer-template duplexusing the polymerase, thereby forming an extended second primer.However, before primer extension can proceed the RNase H enzyme (e.g.RNase HII) cleaves the hybridized primer at the ribobase location. The5′ domain remains hybridized to the template with a 3′OH group availablefor primer extension, while the 3′ domain containing the blocking groupis dispersed into the reaction mixture and does not participate innucleic acid amplification.

In some embodiments, extending the first primer results in formation ofa first extended primer. The first extended primer can include some orall of the sequence of the reverse strand of the template. Optionally,the first extended primer includes a second primer binding site.

In some embodiments, extending the second primer results in formation ofa second extended primer. The second extended primer can include some orall of the sequence of the forward strand of the template. Optionally,the second extended primer includes a first primer binding site.

In some embodiments, the methods are performed without subjecting thedouble stranded nucleic acid template to extreme denaturing conditionsduring the amplifying. For example, the methods can be performed withoutsubjecting the nucleic acid template(s) to temperatures equal to orgreater than the Tm of the template(s) during the amplifying. In someembodiments, the methods can be performed without contacting thetemplate(s) with chemical denaturants such as NaOH, urea, guanidium, andthe like, during the amplifying. In some embodiments, the amplifyingincludes isothermally amplifying.

In some embodiments, the methods are performed without subjecting thenucleic acid template(s) to extreme denaturing conditions during 2, 3,4, 5, 10, 15, 20, 25, and 30 consecutive cycles on the low end of therange and 5, 10, 15, 20, 25, 30, or 50 consecutive cycles on the highend of the range of nucleic acid synthesis. For example, the methods caninclude 2, 3, 4, 5, 10, 15, 20, 25, and 30 consecutive cycles on the lowend of the range and 5, 10, 15, 20, 25, 30, or 50 consecutive cycles onthe high end of the range of nucleic acid synthesis without contactingthe nucleic acid template(s) with a chemical denaturant or raising thetemperature above 50 or 55° C. In some embodiments, the methods caninclude performing 2, 3, 4, 5, 10, 15, 20, 25, and 30 consecutive cycleson the low end of the range and 5, 10, 15, 20, 25, 30, or 50 consecutivecycles on the high end of the range of nucleic acid synthesis withoutsubjecting the nucleic acid template(s) to temperatures that are greaterthan 25, 20, 15, 10, 5, 2 or 1° C. below the actual or calculated Tm ofthe template, or population of templates (or the actual or calculatedaverage Tm of the template, or population of templates). The consecutivecycles of nucleic acid synthesis may or may not include interveningsteps of partial denaturation and/or primer extension.

In some embodiments, the disclosed methods (and related compositions,systems and kits) can further include linking one or more extendedprimer strands to a support. The linking can optionally be performedduring the amplifying, or alternatively after the amplification iscomplete. In some embodiments, the support includes multiple instancesof a second blocked primer, and the methods can include hybridizing atleast one of the extended first primer strands to a second blockedprimer of the support.

In some embodiments, the disclosed methods (and related compositions,systems and kits) can further include linking one or more extendedsecond primer strands to a support. In some embodiments, the support isattached to a first blocked primer. For example, the support can includemultiple instances of a first blocked primer, and the methods caninclude hybridizing at least one of the extended second primers to afirst blocked primer of the support, thereby linking the extended secondprimer to the support. For example, the first primer can hybridize to afirst primer binding site in the extended second primer.

In some embodiments, the support is attached to a second blocked primer.For example, the support can include multiple instances of a secondblocked primer, and the methods can include hybridizing at least one ofthe extended first primers to a second blocked primer of the support,thereby linking the extended first primer to the support. For example,the first blocked primer can hybridize to a second primer binding sitein the extended first primer.

In some embodiments, the support includes both at least one firstblocked primer and at least one second blocked primer, and the disclosedmethods (and related compositions, systems and kits) including linkingboth an extended first primer and an extended second primer to thesupport.

In some embodiments, the support is attached to a target-specificblocked primer. The target-specific primer optionally hybridizes (or iscapable of hybridizing) to a first subset of templates within thereaction mixture, but is unable to bind to a second subset of templateswithin the reaction mixture.

In some embodiments, the support is attached to a universal blockedprimer. The universal primer optionally hybridizes (or is capable ofhybridizing) to all, or substantially all, of the templates within thereaction mixture.

In some embodiments, the reaction mixture includes a first supportcovalently attached to a first target-specific blocked primer and asecond support covalently attached to a second target-specific blockedprimer, and wherein the first and second target-specific primers aredifferent from each other.

In some embodiments, the first target-specific blocked primer issubstantially complementary to a first target nucleic acid sequence andthe second target-specific blocked primer is substantially complementaryto a second target nucleic acid sequence, and wherein the first andsecond target nucleic acid sequences are different.

In some embodiments, the disclosed methods include forming a firstamplicon by amplifying a first template onto a first support, andforming a second amplicon by amplifying a second template onto a secondsupport, optionally within the same continuous phase of a reactionmixture. The first amplicon is optionally linked or attached to thefirst support, and the second amplicon is optionally linked or attachedto the second support.

The disclosed methods optionally comprise producing two or moremonoclonal, or substantially monoclonal, amplicons by clonallyamplifying two or more polynucleotide templates. The two or morepolynucleotide templates are optionally clonally amplified within acontinuous liquid phase of an amplification reaction mixture. Thecontinuous liquid phase of the amplification reaction mixture caninclude a continuous aqueous phase. In some embodiments, the amplifyingincludes generating at least two substantially monoclonal populations ofamplified polynucleotides, each of said populations being formed viaamplification of a single polynucleotide template. In some embodiments,the clonally amplifying includes at least one round of RPA. Optionally,the clonally amplifying includes at least one round of template walking.

In some embodiments, the amplifying optionally includes forming anamplification reaction mixture including a continuous liquid phase. Insome embodiments, the continuous liquid phase is a single continuousaqueous phase. The liquid phase can include two or more polynucleotidetemplates, which can optionally be different from each other. Forexample, the two or more polynucleotide templates can include at leastone nucleic acid sequence that is substantially non-identical, orsubstantially non-complementary, to at least one other polynucleotidetemplate within the amplification reaction mixture.

In some embodiments, the amplifying optionally includes forming anamplification reaction mixture including a single continuous aqueousphase having two or more polynucleotide templates. Amplifying optionallyincludes forming two or more substantially monoclonal nucleic acidpopulations by clonally amplifying the two or more polynucleotidetemplates within the single aqueous phase. Optionally, the clonallyamplifying includes at least one round of RPA. Optionally, the clonallyamplifying includes at least one round of template walking.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems and kits) for amplifying one or morenucleic acid templates, optionally in parallel, using partiallydenaturing conditions. In some embodiments, two or more templates areamplified using such methods, optionally in array format. Optionally,the templates are amplified in bulk in solution prior to distributioninto the array. Alternatively, the templates are first distributed tosites in the array and then amplified in situ at (or within) the sitesof the array.

In some embodiments, the methods can include subjecting adouble-stranded nucleic acid template including a primer binding site onat least one strand to at least one cycle of template-based replicationusing a polymerase.

In some embodiments, the at least one cycle of template-basedreplication includes a partial denaturation step, an annealing step, andan extension step.

In some embodiments, the methods include amplifying the double strandednucleic acid template by subjecting the template to at least twoconsecutive cycles of template-based replication.

In some embodiments, the methods include partially denaturing thetemplate. Optionally, the methods include forming a partially denaturedtemplate including a single stranded region. The partially denaturedtemplate can also include a double stranded region. The single strandedregion can contain the primer binding site.

In some embodiments, the partially denaturing includes contacting thedouble stranded template with a recombinase and a blocked primer. Therecombinase and primer may form part of a nucleoprotein complex, and thepartially denaturing includes contacting the template with the complex.

In some embodiments, the methods include forming a primer-templateduplex by hybridizing a blocked primer to the primer binding site of thesingle stranded region. In some embodiments, the primed templateincludes a double stranded region. Optionally, the double strandedregion does not contain a primer binding site.

In some embodiments, the methods include extending the primer of theprimer-template duplex. Optionally, the methods include forming anextended primer.

In some embodiments, different templates can be clonally amplified ontodifferent discrete supports (e.g., beads or particles) without the needfor compartmentalization prior to amplification. In some embodiments,the templates are partitioned or distributed into emulsions prior toamplifying. Optionally, the templates are distributed into dropletsforming part of a hydrophilic phase of an emulsion having adiscontinuous hydrophilic phase and a continuous hydrophobic phase. Insome embodiments, the emulsion droplets of the hydrophilic phase alsoinclude one or more components necessary to practice RPA and includingthe blocked primers and RNase H enzyme. For example, the emulsiondroplets can include a recombinase. Optionally, the droplets include astrand-displacing polymerase. In some embodiments, the droplets includea support-immobilized blocked primer and/or a solution phase blockedprimer. Optionally, the primer can bind to the template, or to anamplification product thereof. Some suitable emulsion compositions foruse with the disclosed amplification methods can be found, for example,in U.S. Pat. Nos. 7,622,280, 7,601,499 and 7,323,305, incorporated byreference herein in their entireties.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits, wherein the nucleic acidamplification further include sequencing an amplified template, orsequencing an extended primer, (e.g. an extended first primer, orextended second primer). The sequencing can include any suitable methodof sequencing known in the art. In some embodiments, the sequencingincludes sequencing by synthesis or sequencing by electronic detection(e.g., nanopore sequencing). In some embodiments, sequence includesextending a template or amplified template, or extending a sequencingprimer hybridized to a template or amplified template, via nucleotideincorporation by a polymerase. In some embodiments, sequencing includessequencing a template or amplified template that is attached to asupport by contacting the template or extended primer with a sequencingprimer, a polymerase, and at least one type of nucleotide. In someembodiments, the sequencing includes contacting the template, oramplified template, or extended primer, with a sequencing primer, apolymerase and with only one type of nucleotide that does not include anextrinsic label or a chain terminating group.

In some embodiments, the template (or amplified product) can bedeposited, localized, or positioned, to a site. In some embodiments,multiple templates/amplified templates/extended first primers aredeposited or positioned to different sites in an array of sites. In someembodiments, the depositing, positioning or localizing is performedprior to amplification of the template. In some embodiments, thedepositing, positioning or localizing is performed after the amplifying.For example, amplified templates or extended first primers can bedeposited, positioned or localized to different sites of an array.

In some embodiments, the disclosed methods result in the production of aplurality of amplicons, at least some of which amplicons include aclonally amplified nucleic acid population. The clonally amplifiedpopulations produced by the methods of the disclosure can be useful fora variety of purposes. In some embodiments, the disclosed methods (andrelated compositions, systems and kits) optionally include furtheranalysis and/or manipulation of the clonally amplified populations(amplicons). For example, in some embodiments, the numbers of ampliconsexhibiting certain desired characteristics can be detected andoptionally quantified.

In some embodiments, the amplifying is followed by sequencing theamplified product. The amplified product that is sequenced can includean amplicon comprising a substantially monoclonal nucleic acidpopulation. In some embodiments, the disclosed methods include formingor positioning single members of a plurality of amplicons to differentsites. The different sites optionally form part of an array of sites. Insome embodiments, the sites in the array of sites include wells(reaction chambers) on the surface of an isFET array, as describedfurther herein.

In some embodiments, methods of downstream analysis include sequencingat least some of the plurality of amplicons in parallel. Optionally, themultiple templates/amplified templates/extended first primers situatedat different sites of the array are sequenced in parallel.

In some embodiments, the sequencing can include binding a sequencingprimer to the nucleic acids of at least two different amplicons, or atleast two different substantially monoclonal populations.

In some embodiments, the sequencing can include incorporating anucleotide into the sequencing primer using the polymerase. Optionally,the incorporating includes forming at least one nucleotide incorporationbyproduct, including hydrogen ions, protons, pyrophosphate, chargetransfer or heat.

Optionally, the nucleic acid to be sequenced is positioned at a site.The site can include a reaction chamber or well. The site can be part ofan array of similar or identical sites. The array can include atwo-dimensional array of sites on a surface (e.g., of a flowcell,electronic device, transistor chip, reaction chamber, channel, and thelike), or a three-dimensional array of sites within a matrix or othermedium (e.g., solid, semi-solid, liquid, fluid, and the like).

In some embodiments, the site is operatively coupled to a sensor. Themethod can include detecting the nucleotide incorporation using thesensor. Optionally, the site and the sensor are located in an array ofsites coupled to sensors.

In some embodiments, the methods (and related compositions, systems andkits) can include detecting the presence of one or more nucleotideincorporation byproducts at a site of the array, optionally using theFET.

In some embodiments, the methods can include detecting a pH changeoccurring within the at least one reaction chamber, optionally using theFET.

In some embodiments, the amplified nucleic acids can be further analyzed(e.g., sequencing) at the site of distribution without recovering andmoving the amplified products to a different site or surface foranalysis (e.g., sequencing).

In some embodiments, methods of downstream analysis include sequencingat least some of the plurality of amplicons in parallel. Optionally, themultiple templates/amplified templates/extended first primers situatedat different sites of the array are sequenced in parallel.

In some embodiments, the methods (and related compositions, systems andkits) can include depositing, positioning or localizing at least onesubstantially monoclonal population at a site. The site can form part ofan array of sites.

In some embodiments, at least one of the sites includes a reactionchamber, support, particle, microparticle, sphere, bead, filter,flowcell, well, groove, channel reservoir, gel or inner wall of a tube.

In some embodiments the nucleic acid templates can be distributed intothe wells of an isFET array and subsequent amplification of templatesinside the wells of the array, an optional step of downstream analysiscan be performed after the amplification that quantifies the number ofsites or wells that include amplification product. In some embodiments,the products of the nucleic acid amplification reactions can be detectedin order to count the number of sites or wells that include an amplifiedtemplate.

For example, in some embodiments the disclosure relates generally tomethods of nucleic acid analysis, comprising: providing a sampleincluding a first number of polynucleotides; and distributing singlepolynucleotides of the sample into different sites in an array of sites.

In some embodiments, the methods can further include formingsubstantially monoclonal nucleic acid populations by amplifying thesingle polynucleotides within their respective sites.

In some embodiments, the sites remain in fluid communication during theamplifying.

In some embodiments, the amplifying includes partially denaturing thetemplate.

In some embodiments, the amplifying includes subjecting the template topartially denaturing temperatures. In some embodiments, the templateincludes a low-melt sequence including a primer binding site, which isrendered single stranded when the template is subjected to partiallydenaturing temperatures.

In some embodiments, the amplifying includes partially denaturing thetemplate.

In some embodiments, the amplifying includes contacting at least twodifferent templates at two different sites of the array with a singlereaction mixture for nucleic acid amplification.

In some embodiments, the reaction mixture includes a recombinase.

In some embodiments, the reaction mixture includes at least one primerincluding a “drag-tag”.

In some embodiments, the amplifying includes performing at least oneamplification cycle that includes partially denaturing the template,hybridizing a primer to the template, and extending the primer in atemplate-dependent fashion. Optionally, the amplifying includesisothermally amplifying. In some embodiments, the amplifying isperformed under substantially isothermal conditions.

In some embodiments, the percentage of sites containing one or moretemplate molecules is greater than 50% and less than 100%.

In some embodiments, the disclosed methods can further include detectinga change in ion concentration in at least one of the sites as a resultof the at least one amplification cycle.

In some embodiments, the disclosure relates generally to methods fordetection of a target nucleic acid comprising: fractionating a sampleinto a plurality of sample volumes wherein more than 50% of thefractions contain no more than 1 target nucleic acid molecule per samplevolumes; subjecting the plurality of sample volumes to conditions foramplification, wherein the conditions include partially denaturingconditions; detecting a change in ion concentration in a sample volumewherein a target nucleic acid is present; counting the number offractions with an amplified target nucleic acid; and determining thequantity of target nucleic acid in the sample. The change in ionconcentration may be an increase in ion concentration or may be adecrease in ion concentration. In some embodiments, the method mayfurther include combining a sample with bead. In some embodiments, themethod may include loading the sample on a substrate wherein thesubstrate includes at least one well.

In some embodiments, subjecting the target nucleic acids to partiallydenaturing conditions includes contacting the target nucleic acidmolecules in their respective sample volumes with a recombinase and apolymerase under RPA conditions.

In some embodiments, subjecting the target nucleic acids to partiallydenaturing conditions includes subjecting the target nucleic acidmolecules to partially denaturing temperatures.

In some embodiments, the disclosure relates generally to compositions(and relate methods for making and using said compositions) comprisingreagents for amplifying one or more nucleic acid templates in parallelusing partially denaturing conditions.

In some embodiments, the disclosure relates generally to methods forclonally amplifying a population of nucleic acid templates onto apopulation of supports in an amplification reaction solution,comprising: clonally amplifying a first template onto a first nucleicacid template onto a first support according to any of the methodsdisclosed herein, and clonally amplifying a second nucleic acid templateonto a second support according to the same method, wherein bothsupports are included within a single continuous liquid phase during theamplifying.

In some embodiments, a method is provided of generating a localizedclonal population of immobilized clonal amplicons of a single-strandedtemplate sequence using a template-walking method, comprising: (a)attaching the single-stranded template sequence (“template 1”) to animmobilization site (“IS1”), wherein IS1 comprises multiple copies of animmobilized blocked primer (“IS1 primer”) which can hybridizesubstantially to template 1, and template 1 is attached to IS1 byhybridization to an IS1 primer, and (b) amplifying template 1 using IS1primer and a non-immobilized optionally blocked RNase cleavable primer(“SP1 primer”) in solution, wherein amplified strands that arecomplementary to the single-stranded template 1 cannot hybridizesubstantially when single-stranded to primers on IS1, whereinamplification generates a localized clonal population of immobilizedclonal amplicons around the point of initial hybridization of template 1to IS In methods provided in this section that include an IS1 and IS1primer, a polymerase, recombinase, and associated proteins are used topractice to methods, along with at least one blocked RNase cleavableprimer.

Also provided is a method of generating separated and immobilized clonalpopulations of a first template sequence (“template 1”) and a secondtemplate sequence (“template 2”), comprising amplifying the first andsecond template sequence to generate a population of clonal amplicons oftemplate 1 substantially attached to first immobilization site (“IS1”)and not to a second immobilization site (“IS2”), or a population ofclonal amplicons of template 2 substantially attached to IS2 and not toIS1, wherein: (a) both templates and all amplicons are contained withinthe same continuous liquid phase, where the continuous liquid phase isin contact with a first and second immobilization site (respectively,“IS1” and “IS2”), and where IS1 and IS2 are spatially separated, (b)template 1 when in single-stranded form comprises a first subsequence(“T1-FOR”) at one end, and a second subsequence (“T1-REV”) at itsopposite end, (c) template 2 when in single-stranded form comprises afirst subsequence (“T2-FOR”) at one end, and a second subsequence(“T2-REV”) at its opposite end, (d) IS1 comprises multiple copies of animmobilized nucleic acid optionally blocked RNase cleavable primer (“IS1primer”) that can hybridize substantially to T1-FOR and T2-FOR when T1and T2 are single-stranded, (e) IS2 comprises multiple copies of animmobilized optionally blocked RNase cleavable primer (“IS2 primer”)that can hybridize substantially to both T1-FOR and T2-FOR when T1 andT2 are single-stranded, (f) the reverse complement of T1-REV whensingle-stranded cannot hybridize substantially to optionally blockedprimers on IS1, but can hybridize substantially to a non-immobilizedoptionally blocked RNase cleavable primer (“SP1”) in the continuousliquid phase; and (g) the reverse complement of T2-REV whensingle-stranded cannot hybridize substantially to primers on IS2, butcan hybridize substantially to a non-immobilized optionally blockedRNase cleavable primer (“SP2”) in the continuous liquid phase. At leastone of the primers in the methods disclosed in this paragraph is ablocked RNase cleavable primer. In some embodiments, at least one of theimmobilized primers in this paragraph is a blocked RNase cleavableprimer.

In some embodiments, in any method described herein, any nucleic acidthat has dissociated from one immobilization site is capable ofsubstantially hybridizing to both immobilization sites and any movement(e.g., movement by diffusion, convection) of said dissociated nucleicacid to another immobilization site is not substantially retarded in thecontinuous liquid phase.

In some embodiments, in any method described herein, the continuousliquid phase is in simultaneous contact with IS1 and IS2.

In some embodiments, in any method described herein, a first portion ofa template that is bound by an immobilized primer does not overlap witha second portion of the template whose complement is bound by anon-immobilized primer.

In some embodiments, in any method described herein, at least onetemplate to be amplified is generated from an input nucleic acid afterthe nucleic acid is placed in contact with at least one immobilizationsite.

In some embodiments, any method described herein comprising the stepsof: (a) contacting a support comprising immobilized primers with asingle-stranded nucleic acid template, wherein: hybridizing a firstimmobilized primer to a primer-binding sequence (PBS) on the template(b) extending the hybridized first primer in template-dependentextension to form an extended strand that is complementary to thetemplate and at least partially hybridized to the template; (c)partially denaturing the template from the extended complementary strandsuch that at least a portion of the PBS is in single-stranded form(“free portion”); (d) hybridizing the free portion to a non-extended,immobilized second primer (e) extending the second primer intemplate-dependent extension to form an extended strand that iscomplementary to the template (f) optionally, separating the annealedextended immobilized nucleic acid strands from one another. In methodsprovided herein, a polymerase, recombinase, and associated proteins areused to practice to methods, along with at least one blocked RNasecleavable primer.

In some embodiments, during amplification, nucleic acid duplexes areformed comprising a starting template and/or amplified strands; whichduplexes are not subjected during amplification to conditions that wouldcause complete denaturation of a substantial number of duplexes.

In some embodiments, the single-stranded templates are produced bytaking a plurality of input double-stranded or single-stranded nucleicacid sequences to be amplified (which sequence may be known or unknown)and appending or creating a first universal adaptor sequence and asecond universal adaptor sequence onto the ends of at least one inputnucleic acid; wherein said first universal adaptor sequence hybridizesto IS1 primer and/or IS2 primer, and the reverse complement of saidsecond universal adaptor sequence hybridizes to at least onenon-immobilized primer. The adaptors can be double-stranded orsingle-stranded.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, for nucleicacid amplification, comprising multiplex nucleic acid amplification,which includes amplifying within a single reaction mixture differentnucleic acid target sequences from a sample containing a plurality ofdifferent nucleic acid target sequences, the amplifying includinggenerating a plurality of 2-50, or at least fifty different amplifiedtarget sequences (or more) by contacting at least a portion of thesample with a polymerase and a plurality of primers under isothermalamplification conditions.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, for nucleicacid amplification, comprising generating substantially monoclonalnucleic acid populations by re-amplifying the amplicons from themultiplex nucleic acid amplification using a nucleic acid amplificationreaction (e.g., a recombinase).

Optionally, methods for multiplex nucleic acid amplification can furtherinclude a recombinase-mediated nucleic acid amplification method whichincludes re-amplifying at least some of the 2-50 or the at least fiftydifferent amplified target sequences by: (a) forming a reaction mixtureincluding a single continuous liquid phase containing (i) a plurality ofsupports, (ii) at least one of the fifty different amplified targetsequences, (iii) a recombinase and (iv) an RNase H; and (b) subjectingthe reaction mixture to amplification conditions using blocked primersaccording to the invention, thereby generating a plurality of supportsattached to substantially monoclonal nucleic acid populations attachedthereto.

In some embodiments, methods for nucleic acid amplification can beconducted in water-in-oil emulsions that provide compartmentalization.

When conducting a nucleic acid amplification using a plurality ofpolynucleotide templates, clonal amplification using traditionalamplification methods typically relies on techniques such ascompartmentalization of the reaction mixture into segregated portions orcomponents that are not in fluid communication with each other in orderto maintain clonality and prevent cross-contamination of differentamplified populations and to maintain adequate yields of monoclonalamplified product. Using such conventional amplification methods, it istypically not feasible to clonally amplify polynucleotide templateswithin the same reaction mixture without resorting tocompartmentalization or distribution of the reaction mixture intoseparate compartments or vessels, because any polynucleotides within thereaction mixture (including templates and/or amplified products) willtend to migrate randomly through the mixture due to diffusion and/orBrownian motion during such amplification. Such diffusion or migrationtypically increases the incidence of polyclonal amplification and thusvery few, if any, monoclonal populations will be produced.

One suitable technique to reduce the production of polyclonalpopulations in conventional amplification methods uses physical barriersto separate individual amplification reactions into discretecompartments. For example, emulsion amplification uses water-in-oilmicroreactors, where an oil phase includes many separate, i.e.,discontinuous, aqueous reaction compartments. Each compartment serves asan independent amplification reactor, thus the entire emulsion iscapable of supporting many separate amplification reactions in separate(discontinuous) liquid phases in a single reaction vessel (e.g., anEppendorf tube or a well). Similarly, an amplification “master mix” canbe prepared and distributed into separate reaction chambers (e.g., anarray of wells), creating a set of discrete and separate phases, each ofwhich defines a separate amplification reaction. Such separate phasescan be further sealed off from each other prior to amplification. Suchsealing can be useful in preventing cross-contamination between paralleland separate reactions. Exemplary forms of sealing can include use oflids or phase barriers (e.g., mineral oil layer on top of an aqueousreaction) to compartmentalize the PCR reactions into individual anddiscrete compartments, between which transfer of reaction componentsdoes not occur.

Other techniques to prevent cross-contamination and reduce polyclonalityrely on immobilization of one or more reaction components (for example,one or more templates and/or primers) during amplification to preventcross contamination of amplification reaction products and consequentreduction in monoclonality. One such example includes bridgeamplification, where all of the primers required for amplification(e.g., forward and reverse primer) are attached to the surface of amatrix support. In addition to such immobilization, additionalimmobilization components can be included in the reaction mixture. Forexample, the polynucleotide template and/or amplification primers cam besuspended in gels or other matrices during the amplification so as toprevent migration of amplification reaction products from the site ofsynthesis. Such gels and matrices typically require to be removedsubsequently, requiring the use of appropriate “melting” or otherrecovery steps and consequent loss of yield.

In some embodiments, the disclosure provides methods for performingsubstantially clonal amplification of multiple polynucleotide templatesin parallel in a single continuous liquid phase of a reaction mixture,without need for compartmentalization or immobilization of multiplereaction components (e.g., both primers) during amplification. Instead,mixtures of polynucleotide templates in solution can be directlycontacted with amplification reaction components and a suitable surfaceor support having a first primer attached thereto. Other componentsrequired for amplification can be provided in the same continuous liquidphase, including a polymerase, one or more types of nucleotide andoptionally a second primer. In some embodiments, the reaction mixturealso includes a recombinase. Optionally, the reaction mixture furtherincludes at least one agent selected from the group consisting of: adiffusion limiting agent, a sieving agent, and a crowding agent.Examples of amplification mixtures suitable for achieving monoclonalamplification of templates contained in a single continuous liquid phaseare described further herein. Optionally, different templates can beamplified onto different locations on a single surface or support, ordifferent templates can be amplified onto different surfaces ordifferent supports within the same reaction mixture.

In some embodiments, methods for nucleic acid amplification comprisehybridization to the template of additional primers wherein the reactionmixture comprises at least one blocked primer of the invention. Forexample, a second primer can be a reverse amplification primer whichhybridizes to at least a portion of one strand of a polynucleotide. Insome embodiments, a second primer comprises an extendible 3′ end. Insome embodiment, a second primer is not attached to a surface.

In some embodiments, a third primer can be a forward amplificationprimer which hybridizes to at least a portion of one strand of apolynucleotide. In some embodiments, a third primer comprises anextendible 3′ end. In some embodiment, a third primer is not attached toa surface. In some embodiments, a third primer comprises a bindingpartner or affinity moiety (e.g., biotin) for enriching the amplifiednucleic acids.

In some embodiments, primers (e.g., first, second and third primers)comprise single-stranded oligonucleotides.

In some embodiments, at least a portion of a primer can hybridize with aportion of at least one strand of a polynucleotide in the reactionmixture. For example, at least a portion of a primer can hybridize witha nucleic acid adaptor that is joined to one or both ends of thepolynucleotide. In some embodiments, at least a portion of a primer canbe partially or fully complementary to a portion of the polynucleotideor to the nucleic acid adaptor. In some embodiments, the nucleic acidadaptor includes one or more universal sequences, for example universalprimer binding sequences. In some embodiments, a primer can becompatible for use in any type of sequencing platform including chemicaldegradation, chain-termination, sequence-by-synthesis, pyrophosphate,massively parallel, ion-sensitive, and single molecule platforms.

In some embodiments, a primer (e.g., first, second or third primer) canhave a 5′ or 3′ overhang tail (tailed primer) that does not hybridizewith a portion of at least one strand of a polynucleotide in thereaction mixture. Typically, the blocked primers do not have an overhangtail, however when the reaction mixture comprises an additional primerthat is a standard (non-blocked primer) it may comprise an overhangtail. In some embodiments, a tailed primer can be any length, including1-50 or more nucleotides in length.

In some embodiments, nucleic acids that have been amplified according tothe present teachings can be used in any nucleic acid sequencingworkflow, including sequencing by oligonucleotide probe ligation anddetection (e.g., SOLiD™ from Life Technologies, WO 2006/084131),probe-anchor ligation sequencing (e.g., Complete Genomic™ orPolonator™), sequencing-by-synthesis (e.g., Genetic Analyzer and HiSeq™,from Illumina), pyrophosphate sequencing (e.g., Genome Sequencer FLXfrom 454 Life Sciences), ion-sensitive sequencing (e.g., Personal GenomeMachine (PGM™) and Ion Proton™ Sequencer, both from Ion Torrent Systems,Inc.), single molecule sequencing platforms (e.g., HeliScope™ fromHelicos™) and nanopore sequencing via read of individual bases as theypass through the nanopores (e.g. MinION from Oxford NanoporeTechnologies).

In some embodiments, nucleic acid that have been amplified according tothe present teachings can be sequenced by any sequencing method,including sequencing-by-synthesis, ion-based sequencing involving thedetection of sequencing byproducts using field effect transistors (e.g.,FETs and ISFETs), chemical degradation sequencing, ligation-basedsequencing, hybridization sequencing, pyrophosphate detectionsequencing, capillary electrophoresis, gel electrophoresis,next-generation, massively parallel sequencing platforms, sequencingplatforms that detect hydrogen ions or other sequencing by-products, andsingle molecule sequencing platforms. In some embodiments, a sequencingreaction can be conducted using at least one sequencing primer that canhybridize to any portion of the polynucleotide constructs, including anucleic acid adaptor or a target polynucleotide.

In some embodiments, the sequencing can be conducted on a support havinga plurality of sequencing reaction sites arranged in an array on thesupport, where the sequencing reaction sites are capacitively coupled toat least one sensor that detects the presence or a change inconcentration of a nucleotide incorporation byproduct (e.g.,pyrophosphate, hydrogen ion, charge transfer, heat). In someembodiments, the support includes at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, 10⁹ reaction sites, where each site is capacitively coupled to atleast one sensor. In some embodiments, the sensor comprises a fieldeffect transistor, including those described in in U.S. Pat. No.7,948,015 to Rothberg et al.; and Rothberg et al, U.S. PatentPublication No. 2009/0026082, hereby incorporated by reference in theirentireties. Other examples of methods of detecting polymerase-basedextension can be found, for example, in Pourmand et al, Proc. Natl.Acad. Sci., 103: 6466-6470 (2006); Purushothaman et al., IEEE ISCAS,IV-169-172; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); Sakata et al., Angew. Chem. 118:2283-2286 (2006); Esfandyapouret al., U.S. Patent Publication No. 2008/01666727; and Sakurai et al.,Anal. Chem. 64: 1996-1997 (1992). In addition detection may be based ona change in capacitance, impedance or conductivity or voltammetry.

In various exemplary embodiments, the methods, systems, and computerreadable media described herein may advantageously be used to processand/or analyze data and signals obtained from electronic orcharged-based nucleic acid sequencing. In electronic or charged-basedsequencing (such as, pH-based sequencing), a nucleotide incorporationevent may be determined by detecting ions (e.g., hydrogen ions) that aregenerated as natural by-products of polymerase-catalyzed nucleotideextension reactions. This may be used to sequence a sample or templatenucleic acid, which may be a fragment of a nucleic acid sequence ofinterest, for example, and which may be directly or indirectly attachedas a clonal population to a solid support, such as a particle,microparticle, bead, etc. The sample or template nucleic acid may beoperably associated to a primer and polymerase and may be subjected torepeated cycles or “flows” of nucleotide addition (which may be referredto herein as “nucleotide flows” from which nucleotide incorporations mayresult) and washing. The primer may be annealed to the sample ortemplate so that the primer's 3′ end can be extended by a polymerasewhenever nucleotides complementary to the next base in the template areadded. Then, based on the known sequence of nucleotide flows and onmeasured output signals of the chemical sensors indicative of ionconcentration during each nucleotide flow, the identity of the type,sequence and number of nucleotide(s) associated with a sample nucleicacid present in a reaction region coupled to a chemical sensor can bedetermined.

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations can be detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzedextension reactions. In one embodiment, templates, optionally pre-boundto a sequencing primer and/or a polymerase, can be loaded into reactionchambers (such as the microwells disclosed in Rothberg et al, citedherein), after which repeated cycles of nucleotide addition and washingcan be carried out. In some embodiments, such templates can be attachedas clonal populations to a solid support, such as particles, bead, orthe like, and said clonal populations are loaded into reaction chambers.

In another embodiment, the templates, optionally bound to a polymerase,are distributed, deposited or positioned to different sites of thearray. The site of the array includes primers and the methods caninclude hybridizing different templates to the primers within differentsites.

In each addition step of the cycle, the polymerase can extend the primerby incorporating added nucleotide only if the next base in the templateis the complement of the added nucleotide. If there is one complementarybase, there is one incorporation, if two, there are two incorporations,if three, there are three incorporations, and so on. With each suchincorporation there is a hydrogen ion released, and collectively apopulation of templates releasing hydrogen ions changes the local pH ofthe reaction chamber. The production of hydrogen ions is monotonicallyrelated to the number of contiguous complementary bases in the template(as well as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereare a number of contiguous identical complementary bases in the template(i.e. a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, can be proportional tothe number of contiguous identical complementary bases. If the next basein the template is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, an additional stepcan be performed, in which an unbuffered wash solution at apredetermined pH is used to remove the nucleotide of the previous stepin order to prevent misincorporations in later cycles. In someembodiments, the after each step of adding a nucleotide, an additionalstep can be performed wherein the reaction chambers are treated with anucleotide-destroying agent, such as apyrase, to eliminate any residualnucleotides remaining in the chamber, which may result in spuriousextensions in subsequent cycles.

In one exemplary embodiment, different kinds of nucleotides are addedsequentially to the reaction chambers, so that each reaction can beexposed to the different nucleotides one at a time. For example,nucleotides can be added in the following sequence: dATP, dCTP, dGTP,dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposure followed bya wash step. The cycles may be repeated for 50 times, 100 times, 200times, 300 times, 400 times, 500 times, 750 times, or more, depending onthe length of sequence information desired.

In some embodiments, sequencing can be performed according to the userprotocols supplied with the PGM™ or Proton™ sequencer. Example 3provides one exemplary protocol for ion-based sequencing using the IonTorrent PGM™ sequencer (Ion Torrent™ Systems, Thermo Fisher Scientific,CA). In some embodiments, sequencing can be performed according to theuser protocols supplied with the Ion S5 or Ion S5 XL sequencer (IonTorrent™ Systems).

In some embodiments, the disclosure relates generally to methods forsequencing a population of nucleic acid templates, comprising: (a)generating a plurality of amplicons by clonally amplifying a pluralityof nucleic acid templates onto a plurality of surfaces, wherein theamplifying is performed within a single continuous phase of a reactionmixture and wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% of the resulting amplicons are substantially monoclonal ormonoclonal in nature. A sufficient number of substantially monoclonal ormonoclonal amplicons can be produced in a single amplification reactionto generate at least 100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1GB or 2 GB of AQ20 sequencing reads on an Ion Torrent PGM™ 314, 316 or318 sequencer. With respect to related high throughput systems, asufficient number of substantially monoclonal or monoclonal ampliconscan be produced in a single amplification reaction to generate at least100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1 GB, 2 GB, 5 GB, 10 GBor 15 GB of AQ20 sequencing reads on an Ion Torrent Proton, S5 or SSXLsequencer. The term “AQ20 and its variants, as used herein, refers to aparticular method of measuring sequencing accuracy in the Ion TorrentPGM™ sequencer. Accuracy can be measured in terms of the Phred-like Qscore, which measures accuracy on logarithmic scale that: Q10=90%,Q20=99%, Q30=99.9%, Q40=99.99%, and Q50=99.999%. For example, in aparticular sequencing reaction, accuracy metrics can be calculatedeither through prediction algorithms or through actual alignment to aknown reference genome. Predicted quality scores (“Q scores”) can bederived from algorithms that look at the inherent properties of theinput signal and make fairly accurate estimates regarding if a givensingle base included in the sequencing “read” will align. In someembodiments, such predicted quality scores can be useful to filter andremove lower quality reads prior to downstream alignment. In someembodiments, the accuracy can be reported in terms of a Phred-like Qscore that measures accuracy on logarithmic scale such that: Q10=90%,Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99%, and Q50=99.999%. In someembodiments, the data obtained from a given polymerase reaction can befiltered to measure only polymerase reads measuring “N” nucleotides orlonger and having a Q score that passes a certain threshold, e.g., Q10,Q17, Q100 (referred to herein as the “NQ17” score). For example, the100Q20 score can indicate the number of reads obtained from a givenreaction that are at least 100 nucleotides in length and have Q scoresof Q20 (99%) or greater. Similarly, the 200Q20 score can indicate thenumber of reads that are at least 200 nucleotides in length and have Qscores of Q20 (99%) or greater.

In some embodiments, the accuracy can also be calculated based on properalignment using a reference genomic sequence, referred to herein as the“raw” accuracy. This is single pass accuracy, involving measurement ofthe “true” per base error associated with a single read, as opposed toconsensus accuracy, which measures the error rate from the consensussequence which is the result of multiple reads. Raw accuracymeasurements can be reported in terms of “AQ” scores (for alignedquality). In some embodiments, the data obtained from a given polymerasereaction can be filtered to measure only polymerase reads measuring “N”nucleotides or longer having a AQ score that passes a certain threshold,e.g., AQ10, AQ17, AQ100 (referred to herein as the “NAQ17” score). Forexample, the 100AQ20 score can indicate the number of reads obtainedfrom a given polymerase reaction that are at least 100 nucleotides inlength and have AQ scores of AQ20 (99%) or greater. Similarly, the200AQ20 score can indicate the number of reads that are at least 200nucleotides in length and have AQ scores of AQ20 (99%) or greater.

In some embodiments, the present teachings provide systems for nucleicacid amplification, comprising any combination of: beads attached with aplurality of at least one blocked primer of the invention (first primer,second primer, third primer,) polynucleotides, recombinase, recombinaseloading protein, single-stranded binding protein (SSB), polymerase,nucleotides, ATP, RNase H enzyme, phosphocreatine, creatine kinase,hybridization solutions, and/or washing solutions. A system can includeall or some of these components. In some embodiments, systems fornucleic acid amplification can further comprise any combination of:buffers and/or cations (e.g., divalent cations).

In some embodiments, the present teachings provide kits for nucleic acidamplification. In some embodiments, kits include any reagent that can beused for nucleic acid amplification. In some embodiments, kits includeany combination of: beads attached with a plurality of at least oneblocked primer of the invention (first primer, second primer, thirdprimer), polynucleotides, recombinase, recombinase loading protein,single-stranded binding protein (SSB), polymerase, nucleotides, ATP,RNase H enzyme, phosphocreatine, creatine kinase, hybridizationsolutions, washing solutions, buffers and/or cations (e.g., divalentcations). A kit can include all or some of these components.

In some embodiments, the disclosure relates generally to methods,compositions, systems useful for amplifying different nucleic acidtemplates in parallel in a plurality of compartmentalized reactionvolumes, as opposed to amplification within a single continuous liquidphase. For example, the nucleic acid templates can be distributed ordeposited into an array of reaction chambers, or an array of reactionvolumes, such that at least two such chambers or volumes in the arrayeach receive a single nucleic acid template. In some embodiments, aplurality of separate reaction volumes is formed. The reaction chambers(or reaction volumes) can optionally be sealed prior to amplification.In another embodiment, the reaction mixture can be compartmentalized orseparated into a plurality of microreactors dispersed within acontinuous phase of an emulsion, the compartmentalized or separatereaction volumes optionally do not mix or communicate, or are notcapable of mixing or communicating, with each other. In someembodiments, at least some of the reaction chambers (or reactionvolumes) include a recombinase, and optionally a polymerase. Thepolymerase can be a strand-displacing polymerase.

In some embodiments, the disclosure relates generally to compositions,systems, methods, apparatuses and kits for nucleic acid synthesis and/oramplification including emulsions. As used herein, the term “emulsion”includes any composition including a mixture of a first liquid and asecond liquid, wherein the first and second liquids are substantiallyimmiscible with each other. Typically, one of the liquids is hydrophilicwhile the other liquid is hydrophobic. Typically, the emulsion includesa dispersed phase and a continuous phase. For example, the first liquidcan form a dispersed phase that is dispersed in the second liquid, whichforms the continuous phase. The dispersed phase is optionally comprisedpredominantly of the first liquid. The continuous phase is optionallycomprised predominantly of the second liquid. In various embodiments,the same two liquids can form different types of emulsions. For example,in a mixture including both oil and water can form, firstly, anoil-in-water emulsion, where the oil is the dispersed phase, and wateris the dispersion medium. Secondly, they can form a water-in-oilemulsion, where water is the dispersed phase and oil is the externalphase. Multiple emulsions are also possible, including a“water-in-oil-in-water” emulsion and an “oil-in-water-in-oil” emulsion.In some embodiments, the dispersed phase includes one or moremicroreactors in which nucleic acid templates can be individuallyamplified. One or more microreactors can form compartmentalized reactionvolumes in which separate amplification reactions can occur. One exampleof a suitable vehicle for nucleic acid amplification includes awater-in-oil emulsion wherein the water-based phase includes severalaqueous microreactors that are dispersed within an oil phase of anemulsion. In some embodiments, the emulsion can further include anemulsifier or surfactant. The emulsifier or surfactant can be useful instabilizing the emulsion under nucleic acid synthesis conditions.

In some embodiments, the disclosure relates generally to a compositioncomprises an emulsion including a reaction mixture. The emulsion caninclude an aqueous phase. The aqueous phase can be dispersed in acontinuous phase of the emulsion. The aqueous phase can include one ormore microreactors. In some embodiments, the reaction mixture iscontained in a plurality of liquid phase microreactors within a phase ofan emulsion. Optionally, the reaction mixture includes a recombinase.Optionally, the reaction mixture includes a plurality of differentpolynucleotides. Optionally, the reaction mixture includes a pluralityof supports. Optionally, the reaction mixture includes any combinationof a recombinase, a plurality of different polynucleotides and/or aplurality of supports. Optionally, at least one of the supports can beattached to a substantially monoclonal nucleic acid population.

In some embodiments, the disclosure relates generally to a compositioncomprising a reaction mixture, the reaction mixture including (i) aplurality of supports, (ii) a plurality of different polynucleotides and(iii) a recombinase, the reaction mixture contained in a plurality ofliquid phase microreactors in an emulsion.

In some embodiments, the disclosure relates generally to a compositioncomprising a reaction mixture, the reaction mixture including (i) arecombinase and (ii) a plurality of supports, at least one of thesupports being attached to a substantially monoclonal nucleic acidpopulation, wherein the reaction mixture is contained in a plurality ofliquid phase microreactors in an emulsion.

In some embodiments, the disclosure relates generally to a compositioncomprising an emulsion. Optionally, the emulsion comprises a hydrophilicphase and a hydrophobic phase. Optionally, the emulsion comprises ahydrophilic phase dispersed in a hydrophobic phase. Optionally, thehydrophilic phase can include any combination of a plurality ofpolynucleotide templates, a plurality of supports and/or a recombinase.Optionally, the hydrophilic phase can include a plurality ofpolynucleotide templates. Optionally, the hydrophilic phase can includea plurality of supports. Optionally, the hydrophilic phase can include arecombinase.

In some embodiments, a composition comprises an emulsion comprising ahydrophilic phase and a hydrophobic phase, wherein the hydrophilic phaseincludes a plurality of polynucleotide templates, a plurality ofsupports and a recombinase.

In some embodiments, the disclosure relates generally to a compositioncomprises an emulsion including a hydrophilic phase dispersed in ahydrophobic phase. Optionally, the hydrophilic phase includes aplurality of microreactors. Optionally, at least two microreactors ofthe plurality includes a different polynucleotide template. Optionally,the sequences of the different polynucleotide templates is the same ordifferent. Optionally, a first microreactor includes a firstpolynucleotide template and a second microreactor includes a secondpolynucleotide template. Optionally, the first and the secondpolynucleotide templates comprise the same or different sequences.Optionally, at least two microreactors of the plurality includes arecombinase.

In some embodiments, a composition comprises an emulsion including ahydrophilic phase dispersed in a hydrophobic phase, wherein thehydrophilic phase including a plurality of microreactors, at least twomicroreactors of the plurality including a different polynucleotidetemplate and a recombinase.

In some embodiments, the hydrophilic phase includes a plurality ofaqueous microreactors, at least two of the microreactors each includinga different polynucleotide template, a support, and a recombinase.

Optionally, a first microreactor includes a first polynucleotidetemplate and a second microreactor includes a second polynucleotidetemplate. Optionally, the first and the second polynucleotide templatescomprise the same or different sequences.

Optionally, at least one of the plurality of supports is linked to aplurality of first primers (e.g., forward amplification primers).

Optionally, the reaction mixture further includes a plurality of asecond primer (e.g., reverse amplification primers).

In some embodiments, at least one of the plurality of supports furtherincludes a plurality of second primers.

In some embodiments, at least one of the plurality of supports includesa plurality of first and second primers.

In some embodiments, the first and second primers comprise the samesequences.

In some embodiments, the first and second primers comprise differentsequences. In some embodiments, the hydrophilic phase further includes apolymerase.

In some embodiments, the polymerase comprises a strand displacingpolymerase. In some embodiments, the hydrophilic phase includesnucleotides.

In some embodiments, the disclosure relates generally to methods (aswell as associated compositions and systems) for nucleic acid synthesis,comprising: (a) forming a reaction mixture; and (b) subjecting thereaction mixture to amplification conditions. Optionally, the reactionmixture is contained within a hydrophilic phase of an emulsion.Optionally, the emulsion includes a hydrophilic phase and a hydrophobicphase. Optionally, the emulsion comprises a hydrophilic phase dispersedin a hydrophobic phase. Optionally, the reaction mixture contains anycombination of a plurality of supports, a plurality of differentpolynucleotides and/or a recombinase. Optionally, the reaction mixturecontains a plurality of supports. Optionally, the reaction mixturecontains a plurality of different polynucleotides. Optionally, thesequences of the different polynucleotide templates is the same ordifferent. Optionally, a first microreactor includes a firstpolynucleotide template and a second microreactor includes a secondpolynucleotide template. Optionally, the first and the secondpolynucleotide templates comprise the same or different sequences.Optionally, the reaction mixture contains a recombinase. Optionally, theamplification conditions include isothermal or thermo-cyclingtemperature conditions. Optionally, the method further includes formingat least two supports subjecting the emulsion to amplificationconditions results in forming a plurality of supports, wherein at leasttwo of the supports are each independently attached to a substantiallymonoclonal nucleic acid population.

In some embodiments, the disclosure relates generally to methods (aswell as associated compositions and systems) for nucleic acid synthesis,comprising: (a) forming a reaction mixture containing a plurality ofsupports, a plurality of different polynucleotides and a recombinase,the reaction mixture contained within a hydrophilic phase of anemulsion; and (b) subjecting the emulsion including the reaction mixtureto isothermal amplification conditions, thereby generating a pluralityof supports and a substantially monoclonal nucleic acid populationattached thereto.

In some embodiments, the emulsion includes a water-in-oil emulsion. Insome embodiments, the liquid phase microreactors comprise a hydrophilicphase. In some embodiments, the emulsion comprises a hydrophilic phasedispersed in a hydrophobic phase. In some embodiments, the reactionmixture is formed in a single reaction vessel. Optionally, the sequencesof the plurality of different polynucleotide templates is the same ordifferent. Optionally, a first polynucleotide template includes a firstsequence and a second polynucleotide template includes a secondsequence. Optionally, the first and the second polynucleotide templatesequences are the same or different. Optionally, at least one of theplurality of supports is linked to a plurality of first primers (e.g.,forward amplification primers). Optionally, the reaction mixture furtherincludes a plurality of a second primer (e.g., reverse amplificationprimers). In some embodiments, at least one of the plurality of supportsfurther includes a plurality of second primers. In some embodiments, atleast one of the plurality of supports includes a plurality of first andsecond primers. In some embodiments, the first and second primerscomprise the same sequences. In some embodiments, the first and secondprimers comprise different sequences. In some embodiments, the nucleicacid synthesis method further includes recovering from the reactionmixture at least some of the supports attached to substantially nucleicacid monoclonal populations. In some embodiments, the nucleic acidsynthesis method further includes depositing onto a surface at leastsome of the supports attached to the substantially monoclonal nucleicacid populations. In some embodiments, the nucleic acid synthesis methodfurther includes forming an array by depositing onto a surface at leastsome of the supports attached to the substantially monoclonal nucleicacid populations. In some embodiments, the nucleic acid synthesis methodfurther includes sequencing at least one substantially monoclonalnucleic acid population attached to the support. In some embodiments,the support comprises a bead, particle, a planar surface, or an interiorwall of a channel or tube. In some embodiments, the reaction mixturefurther includes a polymerase and a plurality of nucleotides. In someembodiments, the polymerase comprises a strand displacing polymerase.

In some embodiments, methods for nucleic acid synthesis comprise formingan emulsion. Optionally, the emulsion comprises a hydrophilic phase anda hydrophobic phase. Optionally, the emulsion comprises a hydrophilicphase dispersed in a hydrophobic phase. Optionally, the hydrophilicphase includes a plurality of microreactors. Optionally, at least twomicroreactors of the plurality include individual polynucleotidetemplates. Optionally, at least two microreactors of the pluralityinclude a different polynucleotide template. Optionally, a firstmicroreactor includes a first polynucleotide template, and a secondmicroreactor includes a second polynucleotide template. Optionally, thefirst and the second polynucleotide templates have the same or differentsequences. Optionally, at least two microreactors of the pluralityincluding a recombinase.

In some embodiments, the disclosure relates generally to methods (aswell as associated compositions and systems) for nucleic acid synthesis,comprising: forming an emulsion including a hydrophilic phase dispersedin a hydrophobic phase, the hydrophilic phase including a plurality ofmicroreactors, at least two microreactors of the plurality including adifferent polynucleotide template and a recombinase.

In some embodiments, the emulsion includes a water-in-oil emulsion. Insome embodiments, the hydrophilic phase further includes a polymerase.In some embodiments, the polymerase is a strand displacing polymerase.In some embodiments, the hydrophilic phase includes nucleotides. In someembodiments, the emulsion is formed in a single reaction vessel.Optionally, the sequences of the different polynucleotide templates arethe same or different. Optionally, a first microreactor includes a firstpolynucleotide template and a second microreactor includes a secondpolynucleotide template. Optionally, the first and the secondpolynucleotide templates comprise the same or different sequences. Insome embodiments, the at least two microreactors of the pluralityinclude a plurality of supports. Optionally, at least one of theplurality of supports is linked to a plurality of first primers (e.g.,forward amplification primers). Optionally, the reaction mixture furtherincludes a plurality of a second primer (e.g., reverse amplificationprimers). In some embodiments, at least one of the plurality of supportsfurther includes a plurality of second primers. In some embodiments, atleast one of the plurality of supports includes a plurality of first andsecond primers. In some embodiments, the first and second primerscomprise the same sequences. In some embodiments, the first and secondprimers comprise different sequences. In some embodiments, thehydrophilic phase includes a reaction mixture. In some embodiments, thereaction mixture comprises a plurality of polynucleotide templates, aplurality of supports and a recombinase. In some embodiments, methodsfor nucleic acid synthesis further comprise subjecting the emulsion(e.g., including the reaction mixture) to isothermal amplificationconditions, thereby generating a plurality of substantially monoclonalnucleic acid populations. In some embodiments, the plurality ofsubstantially monoclonal nucleic acid populations is attached to theplurality of supports. In some embodiments, the nucleic acid synthesismethod further includes recovering from the reaction mixture at leastsome of the supports attached to substantially nucleic acid monoclonalpopulations. In some embodiments, the nucleic acid synthesis methodfurther includes depositing onto a surface at least some of the supportsattached to the substantially monoclonal nucleic acid populations. Insome embodiments, the nucleic acid synthesis method further includesforming an array by depositing onto a surface at least some of thesupports attached to the substantially monoclonal nucleic acidpopulations. In some embodiments, the nucleic acid synthesis methodfurther includes sequencing at least one substantially monoclonalnucleic acid population attached to the support. In some embodiments,the support comprises a bead, particle, a planar surface, or an interiorwall of a channel or tube. In some embodiments, the reaction mixturefurther includes a polymerase and a plurality of nucleotides. In someembodiments, the polymerase comprises a strand displacing polymerase.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control.

It will be appreciated that there is an implied “about” prior to thetemperatures, concentrations, times, etc., discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”,“containing”, “include”, “includes”, and “including” are not intended tobe limiting.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention.

Unless otherwise defined, scientific and technical terms used inconnection with the present teachings described herein shall have themeanings that are commonly understood by those of ordinary skill in theart. Generally, nomenclatures utilized in connection with, andtechniques of, cell and tissue culture, molecular biology, and proteinand oligo- or polynucleotide chemistry and hybridization describedherein are those well-known and commonly used in the art. Standardtechniques are used, for example, for nucleic acid purification andpreparation, chemical analysis, recombinant nucleic acid, andoligonucleotide synthesis. Enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications oras commonly accomplished in the art or as described herein. Thetechniques and procedures described herein are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the instant specification. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (Third ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques described herein are those well-known and commonly usedin the art.

As utilized in accordance with exemplary embodiments provided herein,the following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “monoclonal” and its variants, when used in reference to one ormore polynucleotide populations, refers to a population ofpolynucleotides where at least 90% of the members of the populationshare at least 90% identity at the nucleotide sequence level. As usedherein, the phrase “substantially monoclonal” and its variants, whenused in reference to one or more polynucleotide populations, refer toone or more polynucleotide populations wherein an amplified templatepolynucleotide molecule is the single largest polynucleotide in thepopulation. Accordingly, all members of a monoclonal or substantiallymonoclonal population need not be completely identical or complementaryto each other. For example, different portions of a polynucleotidetemplate can become amplified or replicated to produce the members ofthe resulting monoclonal population; similarly, a certain number of“errors” and/or incomplete extensions may occur during amplification ofthe original template, thereby generating a monoclonal or substantiallymonoclonal population whose individual members can exhibit sequencevariability amongst themselves. In some embodiments, a low orinsubstantial level of mixing of non-homologous polynucleotides mayoccur during nucleic acid amplification reactions disclosed herein, andthus a substantially monoclonal population may contain a minority ofdiverse polynucleotides (e.g., less than 30%, less than 20%, less than10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, or lessthan 0.001%, of diverse polynucleotides). In certain examples, at least90% of the polynucleotides in the population are at least 90% identicalto the original single template used as a basis for clonal amplificationto produce the substantially monoclonal population. In some embodiments,methods for clonally amplifying provided herein, yield a population ofpolynucleotides wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the members of apopulation of polynucleotides share at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the template nucleic acidfrom which the population was generated. In some embodiments, methodsfor clonally amplifying provided herein, yield a population ofpolynucleotides in which a large enough fraction of the polynucleotidesshare enough sequence identity to allow sequencing of at least a portionof the amplified template using a high throughput sequencing system.

In some embodiments, at least 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,of the members of the amplicon will share greater than 90%, 95%, 97%,99%, or 100% identity with the polynucleotide template. In someembodiments, members of a nucleic acid population produced using methodsprovided herein, can hybridize to each other under high stringencyhybridization conditions.

In some embodiments, methods provided herein generate a population ofpolynucleotides that includes sufficiently few polyclonal contaminantsto be successfully sequenced in a high throughput sequencing method. Forexample, methods provided herein can generate a population ofpolynucleotides that produces a signal (e.g., a sequencing signal, anucleotide incorporation signal and the like) that can be detected usinga particular sequencing system. Optionally, the signal can subsequentlybe analyzed to correctly determine the sequence and/or base identity ofany one or more nucleotides present within any polynucleotide of thepopulation. Examples of suitable sequencing systems for detection and/oranalysis of such signals include the Ion Torrent sequencing systems,such as the Ion Torrent PGM™ sequence systems, including the 314, 316and 318 systems, the Ion Torrent Proton™ sequencing systems, includingProton I, (Life Technologies, Carlsbad, Calif.) and the Ion TorrentProton™ sequencing systems, including Ion S5 and S5XL (Thermo FisherScientific, CA). In some embodiments, the monoclonal amplicon permitsthe accurate sequencing of at least 5 contiguous nucleotide residues onan Ion Torrent sequencing system.

As used herein, the term “clonal amplification” and its variants referto any process whereby a substantially monoclonal polynucleotidepopulation is produced via amplification of a polynucleotide template.In some embodiments of clonal amplification, two or more polynucleotidetemplates are amplified to produce at least two substantially monoclonalpolynucleotide populations.

As used herein, a “blocked primer” or a “3′-blocked primer” cannot beextended by a polymerase. Typically, a 3′-OH is missing or a chemicalmoiety is used to block polymerase extension. For example, the primermay have 3′-phosphate, 3′ biotin, 3′ amine, C3 spacer (3′ Propyl),Spacer 9/18 either at the 3′ end or close to it, 1′,2′-Dideoxyribose(dSpacer), 3′ Hexanediol, 2′-3′-Dideoxy, 3′-deoxy bases, inverted dT(see modified bases and spacers by IDT), 3′-amine or any other moietythat disables polymerase extension, see for example Table 2 in Lin-Linget al. “Single-base discrimination mediated by proofreading inert allelespecific primers”, J. Biochem Mol. Biol. 2005 Jan. 31; 38(1):24-7.

As used herein, a “ribobase” means one or more nucleotides that arecleavable by an RNase H enzyme. The ribobase can be an rU, rA, rC, orrG, as non-limiting examples.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions and kits, for nucleic acid amplification,which includes cleaving with an endonuclease an oligonucleotide that ishybridized to a template nucleic acid. Cleaving the oligonucleotide can,optionally, be followed by an isothermal amplification reaction,especially recombinase-mediated amplification reactions such as RPA(recombinase-polymerase amplification). The methods and compositions usean oligonucleotide including a cleavable moiety, wherein theoligonucleotide is not extendable by a polymerase. That oligonucleotideis also referred to herein as a “blocked primer”, wherein the cleavablemoiety separates a 5′ domain and 3′ domain of the primer, and wherein anendonuclease, such as RNase H as disclosed herein, cleaves the primer atthe cleavable moiety location removing the block. The 5′ domain of theoligonucleotide remains hybridized to the template nucleic acid afterthe block is removed. Accordingly, the endonuclease cleaves orhydrolyzes a location or residue on the oligonucleotide when it ishybridized to a nucleic acid template. In some embodiments, theoligonucleotide and template nucleic acid form a DNA:DNA duplex. Infurther embodiments, the endonuclease does not cleave theoligonucleotide or blocked primer at other nucleotide positions besidesthe cleavable moiety.

In some embodiments, the cleavable moiety is one or more nucleotidesthat are cleavable by an RNase H enzyme, wherein the endonuclease isRNase H. Nucleotides cleavable by RNase H include ribobases rU, rA, rC,and rG, any of which may be present as a single ribobase or as two ormore at the desired cleavage location in the oligonucleotide or blockedprimer. In some embodiments, the RNase H enzyme is any RNase H enzymedisclosed herein, and is typically active at 37° C. and is other than athermostable enzyme. In some embodiments, the RNase H enzyme is RNaseII.

In some embodiments, the cleavable moiety is a site cleavable by anapurinic/apyrimidinic (AP) endonuclease when the oligonucleotide orblocked primer is hybridized to the nucleic acid template forming adouble stranded duplex. An abasic, or baseless, site is cleavable by APendonucleases and includes an apurinic site, an apyrimidinic site or aspacer. In some embodiments, AP endonucleases include Endonuclease IV(Endo IV), APE 1 or APE 2. See Example 6. In some embodiments, theendonuclease, including AP endonucleases, is an enzyme that is active at37° C. and/or is other than a thermostable endonuclease.

The oligonucleotide or primer configuration can be any of thosedisclosed herein as to length of the 5′ domain, length of the 3′ domainand/or location of the cleavable moiety. In some embodiments, the firstoligonucleotide or blocked primer is between 15 and 200, 15 and 150, 15and 100, or 15 and 50 nucleotides long, and wherein the cleavable moietyis more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides awayfrom the 3′ terminus of the oligonucleotide. In some embodiments, theblocked oligonucleotide primer (e.g. first or second oligonucleotide;forward or reverse primer) includes a 5′ domain and a 3′ domainseparated by the one or more nucleotides that are cleavable by RNase H,wherein the 5′ domain is 10 to 50 nucleotides in length and the 3′domain is 10 to 50 nucleotides in length. In some embodiments, the RNaseH enzyme is RNase 1-111.

In some embodiments, the first oligonucleotide is between 15 and 200nucleotides long, and wherein the abasic site or spacer is more than 5nucleotides away from the 3′ terminus of the oligonucleotide. In someembodiments, the blocked oligonucleotide primer includes a 5′ domain anda 3′ domain separated by an abasic site or spacer that is cleavable byan AP endonuclease, wherein the 5′ domain is 10 to 50 nucleotides inlength and the 3′ domain is 10 to 50 nucleotides in length. In someembodiments, the AP endonuclease is Endo IV or APE 1.

In some embodiments, the first oligonucleotide is a universal primer,wherein the universal primer can include any of the primerconfigurations disclosed herein. In some embodiments, the method ofcleaving a hybridized oligonucleotide includes the use of both a firstand second oligonucleotide, e.g. a forward and reverse primer that bindto complementary strands of the template nucleic acid in reverseorientation. In some embodiments, the reaction mixture further includesa second oligonucleotide that binds to the target nucleic acid on acomplementary strand to the first binding site and in a reverseorientation, and wherein the second oligonucleotide is not extendable bythe polymerase.

In some embodiments, the method of cleaving a double stranded templatenucleic acid is carried out according to the following steps: forming areaction mixture comprising the template nucleic acid and a firstoligonucleotide comprising a cleavable moiety, wherein the firstoligonucleotide is not extendable by a polymerase; exposing the reactionmixture to an endonuclease enzyme in the presence of reaction conditionspermissive for hybridization between the first oligonucleotide and afirst binding site on the template nucleic acid, wherein at least aportion of the first oligonucleotide hybridizes to the template; andcleaving the first oligonucleotide at the cleavable moiety with theendonuclease, wherein the endonuclease cleaves the oligonucleotide moreefficiently at 37° C. than at 60° C., wherein the cleavage step occursat a temperature below 42° C., or a combination thereof.

In some embodiments, the endonuclease cleaves a significant fraction ofthe first oligonucleotide present in the reaction mixture within 30 or60 minutes of exposing the hybridized primer and template nucleic acidto the endonuclease. To determine whether the endonuclease cleaves asignificant fraction of oligonucleotides present, as a non-limitingexample, the oligonucleotides before cleavage can be non-extendable by apolymerase wherein when the oligonucleotide is cleaved extension canoccur and a product of the extension can be detected. In someembodiments, the nucleic acid template is a member of a polynucleotidelibrary and the method is carried out on a plurality of nucleic acidtemplates of the polynucleotide library, wherein each member of thepolynucleotide library comprises the first primer binding sequence.Accordingly, the method can be combined with amplification methods, suchas RPA, wherein the reaction mixture further comprises recombinase andpolymerase enzymes to amplify the target nucleic acid. The recombinaseand polymerase enzyme can be any of those disclosed herein, and aretypically other than thermostable enzymes. In some embodiments, thereaction mixture remains below 42° C. for both the cleavage andamplification. Accordingly, the cleavage and amplification are carriedat a temperature below 42° C.

In some embodiments, the first primer is a universal primer, thereaction mixture is subject to amplification conditions, and at leasttwo of the nucleic acid templates are amplified to form substantiallymonoclonal populations. The first primer can be immobilized on a solidsupport or the amplification method comprises bridge amplification oremulsion amplification. In some embodiments, the amplification iscarried out for at least 10 cycles using the first oligonucleotide andthe second oligonucleotide. In some embodiments, the cleavage and 10cycles are carried out in less than 15, 30, or 60 minutes.

In some embodiments, provided herein are reaction mixture compositions,wherein the reaction mixture comprises a population of cleavableprimers, wherein the population of cleavable primers comprises at least10 primers that bind to at least 10 target binding sites on a mammaliangenome, wherein the cleavable primers comprise a 5′ domain and a 3′domain separated by one or more nucleotides that are cleavable by anRNase H, wherein the 5′ domain is 10 to 50 nucleotides in length and the3′ domain is 10 to 50 nucleotides in length. In alternative embodiments,the reaction mixture comprises a population of cleavable primers,wherein the population of cleavable primers comprises at least 10primers that bind to at least 10 target binding sites on a mammaliangenome, wherein the cleavable primers comprise a 5′ domain and a 3′domain separated by an abasic site or spacer that is cleavable by an APendonuclease, wherein the 5′ domain is 10 to 50 nucleotides in lengthand the 3′ domain is 10 to 50 nucleotides in length. In someembodiments, the 3′ domain of the cleavable primer is 14 to 25nucleotides in length.

The reaction mixture compositions can further include a polymerase, arecombinase or both. Those enzymes can be any of those disclosed hereinand are typically active at 37° C. and/or other than thermostableenzymes. In some embodiments, the population comprises at least 100cleavable primers that are not extendable by a polymerase or wherein thepopulation comprises at least 1000 cleavable primers that are notextendable by a polymerase.

Those skilled in the art can devise many modifications and otherembodiments within the scope and spirit of the disclosed inventions.Indeed, variations in the materials, methods, drawings, experimentsexamples and embodiments described may be made by skilled artisanswithout changing the fundamental aspects of the disclosed inventions.Any of the disclosed embodiments can be used in combination with anyother disclosed embodiment.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how touse the embodiments provided herein, and are not intended to limit thescope of the disclosure nor are they intended to represent that theExamples below are all of the experiments or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by volume, and temperature is in degreesCentigrade. It should be understood that variations in the methods asdescribed can be made without changing the fundamental aspects that theExamples are meant to illustrate.

Example 1: Screening of RNase H Cleavable 3′ Blocked PrimerConfigurations for Use in a Recombinase Polymerase AmplificationReaction

Experiments were performed that analyzed configurations for blockedforward primers and blocked reverse primers for use in methods forperforming recombinase polymerase amplification of a nucleic acidtemplate under isothermal amplification conditions. Configurationsanalyzed in these experiments included the length of the 3′ domain ofthe blocked forward and reverse primers and the identity of theribobase, which is the site of RNase H cleavage located between the 5′domain and 3′ domain of the 3′ blocked primers. See FIG. 2 for a diagramof the 3′ blocked primers and the different configurations analyzed inthese Examples.

Recombinase polymerase amplification (RPA) reactions were performedusing the following protocol in a single reaction vessel with a singlecontinuous liquid phase in a total reaction volume of ˜50 μL:

The recombinase source was from a TwistAmp Basic kit (TwistDx,Cambridge, Great Britain). Dehydrated pellets in the kit contain uvsXrecombinase, uvsY recombinase loading protein, gp32 protein, Sau DNApolymerase, dNTPs, ATP, phosphocreatine and creatine kinase. One pelletfrom a TwistAmp Basic kit was rehydrated in 29.5 uL of Rehydrationbuffer supplied from the kit in a 0.2 μL PCR tube. The recombinasesolution was vortexed and spun, then iced.

The template DNA (at various concentrations) in a volume of 0.5 μL wasadded to the reaction tube, vortexed and then spun. The template DNA waseither a genomic DNA, or a DNA library made with adapters foramplification.

The 3′ blocked primers with a single ribobase were synthesized by IDT(Coralville, Iowa) in various configurations of long and short 5′ and 3′domains designated as V1-V6. See FIG. 2 and FIG. 10. A long 5′ domain isgreater than 25 nucleotides in length and a long 3′ domain is equal to,or greater than 14 nucleotides in length, whereas a short 5′ domain is15-25 nucleotides in length, and a short 3′ domain is 4-6 nucleotides inlength. The medium length 3′ domain tested was 10 nucleotides in length.In these experiments the 3′ blocking group was a 3′-C3 spacer, but it isunderstood that other 3′-blocking groups such as a phosphate, biotin,amine, etc. can be used with the 3′ blocked primer.

Forward and reverse primers (various amount of 10 uM stocks) and RNaseHII (NEB, Ipswich, Mass.; various unit amount) were added to thereaction tube. Additional low-TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH8.0) was added to top off the total volume to 42.5 μL. The recombinasereaction mix was vortexed, spun, and placed on ice. Next, 7.5 μL of 54mM Mg-acetate was added to the inside lid surface of the PCR tubecontaining the recombinase reaction. The PCR tube was closed, thenvortexed and spun, and incubated in a total 50 μL volume at 37° C., for30 min to 50 minutes in the thermocycler. The reaction was stopped byadding EDTA at 20 mM final concentration, then purified with PureLink®PCR Purification kit (Thermo Fisher Scientific, Waltham, Mass.).Amplified product was eluted in 50 μL elution buffer supplied in thekit. A portion of the product was assessed on E-Gel® EX Agarose Gels, 2%(Thermo Fisher Scientific, Waltham, Mass.) containing a nucleic acidstain, such as SYBRGOLD.

Two separate experiments were conducted to determine the optimal 5′ and3′ domain length as well as the choice of ribobase for the RPA reaction.The product from the above RPA reaction was obtained using 1 pM oftemplate (100 bp insert library with a tailed-A (57 bp) and P1B (53 bp)adapters with an expected amplicon size of about 210 bp), 400 nm each of3′ blocked primers and 8 mM of Mg-acetate, wherein 15 μL of the 504,purified RPA reaction product was loaded into wells of the agarose gelsand the DNA fragments separated by electrophoresis. See FIGS. 3-5 andTables 1-2.

Table 1 provides the tested primer designs wherein V1 (short 5′ domainand short 3′ domain), V2 (long 5′ domain and long 3′ domain) and V3(long 5′ domain and short 3′ domain) correspond to the 3′ blockedprimers of FIG. 2 and the ribobase is cysteine (rC) or uracil (rU). MMis a mismatched base following the 3′ domain. See FIG. 3 for the gelthat corresponds to Table 1.

TABLE 1 Configurations for screening V1, V2 and V3 primer designs LanePrimer configuration Results M NEB Low MW ladder N/A 1 Regular short (20mer) Significant primer dimers (nonspecific primers bands) 2 Regularlong (46-53 mer) Significant primer dimers (nonspecific primers bands) 33′ blocked primers. V1-rC No detectable results 4 3′ blocked primers.V2-rC Amplification band present with no primer dimers 5 3′ blockedprimers. Amplification band present with no V2-MM-rC primer dimers 6 3′blocked primers. V2-rU Amplification band present with no primer dimers7 3′ blocked primers. V3 No detectable product band

The results from the use of the V2 primer configuration indicated thechoice of ribobase is not critical and that the presence of a 3′mismatch base is optional. In comparing the primer configurations of V1,V2 and V3 the results indicated a short (4-6 nucleotides) 3′ domain wasinefficient in amplifying nucleic acid under the RPA reaction conditionsanalyzed in this experiment, with either a long or short 5′ domain.However, surprisingly, a relatively long (≥14 nucleotides) 3′ domainwith a long 5′ domain resulted in efficient amplification of templateDNA. Accordingly, in some embodiments, the methods for amplifying anucleic acid template provided herein, include a 3′ blocked primer thatincludes a 5′ domain and a 3′ domain separated by a ribobase, whereinthe 3′ domain is ≥14 nucleotides in length, such as 14 to 30 nucleotidesin length.

To further elucidate the configuration of 5′ and 3′ domain length, acomparison between V4 (long 5′ domain and medium 3′ domain) and V5(short 5′ domain and long 3′ domain) was performed. Table 2 provides thetested primers wherein V4 and V5 correspond to the 3′ blocked primers ofFIG. 2 and the ribobase is guanine (rG) or uracil (rU). See FIGS. 4 and5 for the gels that corresponds to Table 2; the gel of FIG. 5 wasexposed for a longer period of time to the detection reagent resultingin the detection of a weak product band in lane 3 (V4-rU).

TABLE 2 Primer configuration screening for V4 and V5 as compared toV2-rU Lane Primer configuration Results M NEB Low MW ladder N/A 1 3′blocked primers. Amplification band present with no primer V2-rU dimers2 3′ blocked primers. Amplification band present with no primer V5-rG/rUdimers 3 3′ blocked primers. Weak (FIG. 5) or no detectable (FIG. 4)V4-rU amplification product band with no primer dimers

The results of the comparison between V4 and V5 indicated: 1) due to thepresence of a weak band in FIG. 5 for V4, that there was someamplification where the 3′ domain of the 3′ blocked primer had a lengthof 10 nucleotides, in embodiments less than 14 nucleotides but more than6 nucleotides; and, 2) a long 3′ domain (≥14 nucleotides) efficientlyamplified DNA in the RPA reaction with a 5′ domain of 15 nucleotides orgreater. Accordingly, in some embodiments, the methods for amplifying anucleic acid template provided herein, include a 3′ blocked primer thatincludes a 5′ domain and a 3′ domain separated by a ribobase, whereinthe length of the 3′ domain is equal to or greater than 7, 8, 9, 10, 11,12, 13 or 14 nucleotides on the low end of the range and equal to orless than 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides on the high end of the range. In embodiments the length ofthe 3′ domain is from about 7 to 25, about 10 to 25 or about 14 to 25nucleotides and the length of 5′ domain is from about 15 to 60, about 15to 40 or about 15 to 25 nucleotides.

The results of these experiments indicated efficient amplification using3′ blocked primers with a 5′ DNA domain equal to or greater than (≥)15nt and 3′ DNA domain equal to or greater than (≥) 14nt (excluding anypossible 3′ mismatched nucleotides). The cleaved 3′ domain by RNase Henzyme is greater than or equal to (≥) 15 nucleotides when including theribobase in the length. The ribobases uracil, guanine and adenine areapproximately equivalent but the interaction between RNase H enzyme andcysteine ribobase may result in slower kinetics under certain reactionconditions. In the experiments described herein, the primers V2 and V5produced amplification product with no detectable nonspecificamplification. Accordingly, in some embodiments, the methods foramplifying a nucleic acid template provided herein, include a 3′ blockedprimer that includes a 5′ domain and a 3′ domain separated by aribobase, wherein the 5′ domain is at least 25 nucleotides in length andthe 3′ domain is at least 14 nucleotides in length. In some embodiments,the 5′ domain of the 3′ blocked primer is 15-25 nucleotides in lengthand the 3′ domain is at least 14 nucleotides in length. In someembodiments, the length of 5′ domain is at least 15 nucleotides and thelength of the 3′ domain is at least 14 nucleotides. In yet otherembodiments, the methods for amplifying a nucleic acid template providedherein, include a 3′ blocked primer that includes a 5′ domain and a 3′domain separated by a ribobase, wherein the 5′ domain is 15 to 50nucleotides in length and the 3′ domain is 14 to 50 nucleotides inlength.

Example 2: Use of RNase H Cleavable 3′ Blocked Primer in a RecombinasePolymerase Amplification Reaction Reduces Nonspecific Product Formation

Experiments were performed that analyzed the efficiency of the blockedforward primers and blocked reverse primers for use in methods forperforming recombinase polymerase amplification of a nucleic acidtemplate under isothermal amplification conditions as compared tostandard (non-blocked) primers and the amplification of nonspecific DNAsuch as primer dimers. The results illustrate that the cleavable 3′blocked primers provided herein reduce or eliminate primer dimerformation in RPA methods.

First, 3′ blocked primers (forward and reverse) demonstrated an abilityto reduce nonspecific product amplification in a control reactionperformed in the absence of template DNA, as compared to standard(non-blocked) short and long primers. The RPA reaction was carried outas described in Example 1 except that no template was included in thereaction mixture, which included 400 nm each of 3′ blocked primers, withor without RNase HII enzyme, and 8 mM of Mg-acetate, wherein 15 μL ofthe 504, purified RPA reaction product was loaded into wells of theagarose gel and the DNA fragments separated by electrophoresis. See FIG.6 and Table 3.

Non-specific amplification was tested in a no template control reaction.3′ blocked primers (V2-rU design) were compared to standard(non-blocked) long and short primers and V2-rU (long 5′ domain and long3′ domain) (Table 3). The results are shown on FIG. 6. Regular(unblocked) primers produced significant amounts of spuriousamplification products in the no template control reactions. On theother hand, using the V2-rU 3′ blocked primers, only minimal nonspecificproducts were detected.

TABLE 3 Reduced non-speific amplification product with V2-rU as comparedto non-blocked primers Lane Primer configuration Results M NEB Low MWladder N/A 8 NTC: Standard short primers Nonspecific amplificationproduct present 9 NTC: Standard long primers Nonspecific amplificationproduct present 10 NTC: 3′ blocked primers Minimal nonspecificamplification V2-rU product

Next, the formation of nonspecific products such as primer dimers wasanalyzed with the use of 3′ blocked primers compared to standard(non-blocked) primers wherein the 3′ blocked primers were designed tohave functional primers after cleavage (by RNase H enzyme) that matchand have the same sequence as the standard primers used in the RPAreaction. Recombinase polymerase amplification (RPA) was performed using100 ng of template (E. coli genomic DNA), 480 nm each of blocked andstandard primers (SEQ ID Nos:2-5), RNase H enzyme and 8 mM ofMg-acetate, wherein 5 or 15 μL of the 500_, purified RPA reactionproduct was loaded into wells of the agarose gel, and the DNA fragmentswere separated by electrophoresis. See FIG. 7 and Table 4.

The following standard (non-blocked) primers were used:

268-Forward primer: (SEQ ID NO: 2)ACA CGG TCC ADA CTC CTA CGG GAG GCA GCA(e.g., where according to IUPAC “D” isselected at random from A, T or G). 268-Reverse primer: (SEQ ID NO: 3)GCG GCT GCT GGC ACG GAG TTA GCC GGT GCT

The following 3′ blocked primers, wherein the underlined sequence isidentical to the corresponding forward or reverse standard (non-blocked)primer after cleavage, were used:

268-Forward primer-rG: (SEQ ID NO: 4)ACA CGG TCC ADA CTC CTA CGG GAG GCA GCA rGTG GGG AAT ATT GCA C-block268-Reverse primer-rU: (SEQ ID NO: 5)GCG GCT GCT GGC ACG GAG TTA GCC GGT GCT rUCT TCT GCG GGT AAC G-block

The following E. coli amplicon 268 was generated from template gDNA (202bp) as a result of the RPA reaction:

(SEQ ID NO: 1) ACA CGG TCC AGA CTC CTA CGG GAG GCA GCA GTG GGGAAT ATT GCA CAA TGG GCG CAA GCC TGA TGC AGC CATGCC GCG TGT ATG AAG AAG GCC TTC GGG TTG TAA AGTACT TTC AGC GGG GAG GAA GGG AGT AAA GTT AAT ACCTTT GCT CAT TGA CGT TAC CCG CAG AAG AAG CAC CGGCTA ACT CCG TGC CAG CAG CCG C

Table 4 provides the tested primers, standard primers and 3′ blockedprimers wherein the 5′ domain was 30 nucleotides in length and the 3′domain was 15 nucleotides in length. Each RPA product was loaded at 5 μland 15 μl in two wells, respectively. See FIG. 7 for the gel thatcorresponds to Table 4.

TABLE 4 3′ Blocked primers eliminate primer dimer product in gDNAamplification as compared to standard (non-blocked) primers. Lane Primer& Exp. Configuration Results M NEB low molecular weight N/A ladder 1Standard primers; 5 μL Primer dimer product and loaded amplificationproduct present 2 3′ blocked primers; with No detectable primer dimerRNaseH enzyme; 5 μL product; amplification product loaded present 3 3′blocked primers; No No detectable primer dimer RNaseH enzyme control;product or amplification product 5 μL loaded 4 Standard primers; 15 μLPrimer dimer product and loaded amplification product present 5 3′blocked primers; with No detectable primer dimer RNaseH enzyme; 15 μLproduct; amplification product loaded present 6 3′ blocked primers; NoNo detectable primer dimer RNaseH enzyme control; product oramplification product 15 μL loaded

As shown in FIG. 7, amplification using standard (non-blocked) RPAprimers under these conditions, generated detectable primer dimers. Onthe other hand, primer dimers were not detectable after amplificationusing the 3′ blocked primers of the invention, having a 5′ domain aftercleavage, that contains the same sequence as the standard primers.Furthermore, primer dimers were not detected when the 3′ blocked primerswere uncleaved (no RNase H enzyme), and therefore fail to producedetectable amplification product. Accordingly, this experimentdemonstrates that the blocked forward primers and blocked reverseprimers when used in methods for performing recombinase polymeraseamplification of a nucleic acid template under isothermal amplificationconditions efficiently amplify template DNA without substantiallygenerating nonspecific product such as primer dimer product.

Example 3: Titration of RNase H Enzyme for Use in a RecombinasePolymerase Amplification Reaction with RNase H Cleavable 3′ BlockedPrimers

Experiments were performed that analyzed various amounts of RNase Henzyme, including a “prohibiting” amount, a “limiting” amount and an“excess” amount for use in the methods for performing recombinasepolymerase amplification of a nucleic acid template under isothermalamplification conditions with the blocked forward primers and blockedreverse primers. Not to be limited by theory, in the initial stage ofamplification where low copies of template DNA is present,primer-template duplex “D-loop” is considered a rare event. A certainamount of RNase H enzyme is required to cleave the duplex to activatethe primer before the D-loop dissociates. If RNase H enzyme is too low,the chance of an RNase enzyme interacting with a blocked primer bound toa template may become mathematically impossible so that amplificationdoes not progress. In that instance, the RNase H enzyme is considered tobe a “prohibiting” amount. When more RNase H enzyme is present, cleavageof the template-bound blocked primer becomes possible and moreefficient, while still not fully efficient. In that instance,amplification progresses at a reduced rate, although such anamplification reaction can still achieve sufficient amplificationproduct if allowed a longer reaction time. In that instance, the RNase Henzyme is considered a “limiting” amount. An “excess” amount of theRNase H enzyme ensures the RPA reaction proceeds based on the kineticsof the polymerase and other components in the reaction mixture and notthe enzyme needed to remove the blocking group, which would otherwise bea rate limiting step for primer extension. In that instance, RNase Henzyme is present in an amount to guarantee full cleavage of theribobase-containing primer, and is “non-limiting”; RNase H enzyme willnot slow down or diminish amplification as compared to a “prohibiting”or “limiting” amount of enzyme. In some embodiments, the RNase H enzymeis present in the RPA reaction mixture in an excess amount. Thetitration experiments described herein empirically determine an excessamount of RNase HII enzyme with the V2 and V5 3′ blocked primerconfigurations of the invention in the RPA methods for amplifying DNAtemplate.

The RPA experiment was conducted following the methods of Example 1using template library DNA (100 bp insert library with a tailed-A (57bp) and P1B (53 bp) adapters with an expected amplicon size of about 210bp) and 400 nm each of 3′ blocked primers (V2-rU). The amplificationreaction was performed at 37° C. for 50 minutes. The RNase HII wasconcentrated 20× from the original 5 U/uL concentration. 100 U in a 1 uLvolume (20× concentrate) was tested as compared to 5-20 U in a 1-4 uLvolume (standard concentration from NEB).

FIG. 8A provides the identity of samples loaded onto gels the testedRNase H enzyme and the 3′ blocked primers (V2-rU) wherein the 5′ domainis 25 nucleotides in length and the 3′ domain is 14 nucleotides inlength. Each RPA product was loaded at 150 in a wells of the gel. Asshown in FIG. 8A, when amplified for 50 minutes, as low as 5 U of RNaseHII produced detectable product with exemplified V2-rU 3′ blocked primerconfiguration. All concentrations of RNase HII enzyme used produceddetectable product, with increasing amounts of RNase HII enzyme yieldingmore product, as seen with brighter product bands, however the RNase Henzyme is saturated at 20 U, i.e. an excess amount. RNase H enzyme above20 U, including up to 200 U had no detrimental effect on the productproduced. In the control lane with 3′ blocked primers and no RNase HIIenzyme, no product was detected. Of note, in the control lane withstandard non-blocked primers (and no RNase HII) non-specific productbands were detected confirming results observed in Example 2.

In this experiment, based on the intensity of the product bands, 5-10 Uof RNase HII enzyme is considered limiting for the V2 primerconfiguration in a 50 minute reaction while 20 U-200 U is considered inexcess.

Titration of RNase H enzyme was further analyzed with primerconfigurations V2-rU, V2-rC and V5. FIG. 8B provides the identity ofsamples loaded onto gels, the tested RNase H enzyme and the type of 3′blocked primers. Each RPA product was loaded at 10 μl in wells of thegel. As shown in FIG. 8B, when amplified for 50 minutes, as low as 5 Uof RNase HII produced detectable product with exemplified V2-rU andV2-rC 3′ blocked primer configuration (lanes 3 and 6, respectively) butis “prohibiting” at 2.5 U wherein no detectible product band wasgenerated (lanes 2, 5 and 8). In this experiment, 5-10 U of RNase HIIenzyme is considered “limiting” for the V2 primer configuration in a 50minute reaction based on the intensity of the product bands.

While relatively less efficient under other limiting reaction conditions(e.g. shorter reaction time), ribobase rC (lanes 5-7) performedsimilarly to rU primers (lanes 2-4) in V2 configurations in amount ofproduct generated. Accordingly, in some embodiments the ribobase in the3′ blocked primers can be rC. The V5 primer with short 5′ domain (lanes8-9) is less efficient as compared to V2, and may require more RNase HIIto achieve a similar level of amplification under similar reactioncondition. In this experiment, 2.5-5 U of RNase H enzyme is considered“prohibiting” and 10 U is considered “limiting” for V5 primerconfiguration in a 50 minute reaction based on the intensity of theproduct bands.

For the control (lane 1) of non-blocked standard primers with no RNaseHII enzyme, significant non-specific product bands were observed.

RNase H enzyme was further analyzed at different amplification reactiontemperatures to determine an optimal temperature range for RNase HIIenzyme.

The RPA experiment was conducted following the methods of Example 1using 400 nm each of 3′ blocked primers (V2-rU) and 10 U in a 2 μLvolume of RNase HII from NEB, wherein the reaction was incubated from arange of 37° C. to 42° C. for 50 minutes. Each RPA product was loaded at5 μl in a wells of the gel. FIG. 8C provides the identity of samplesloaded onto gels, the tested RNase H enzyme, the 3′ blocked primers andreaction temperature. As shown in FIG. 8C, RNase HII performs optimallyat 37° C., with a faint band present in lane 3 (40° C. reactiontemperature) and no product band visible in lane 4 (42° C. reactiontemperature). As will be appreciated, different RNase H enzymes mayperform optimally at different temperatures and such conditions may beempirically determined with the 3′ blocked primers in the RPAamplification methods.

Example 4: Use of Alternative Reaction Mixture in Recombinase PolymeraseAmplification Reaction with RNase H Cleavable 3′ Blocked Primers

RPA experiments were performed that analyzed an exemplary alternativebase RPA reaction mixture formulation as compared to the base reactionmixture in the Examples above, which were formed by hydrating acommercially available pellet (TwistDx, Cambridge, Great Britain),adding blocked forward and reverse primers, RNase HII, additional low-TEbuffer, and Mg-acetate.

Reaction mixtures prepared using commercially available pellets fromTwistDx contain uvsX recombinase, uvsY recombinase loading protein, gp32protein, Sau DNA polymerase, dNTPs, ATP, phosphocreatine and creatinekinase. An exemplary alternative reaction mixture was prepared in pelletform, that included the same components as the commercially availablereaction mixture above except that Sau polymerase was replaced with amixture of Sau polymerase and a T7 DNA polymerase, with thioredoxin.After the reaction mixture pellet was hydrated, RNase H enzyme, and 3′blocked primers were added. Except for the reaction mixturemodifications noted, the RPA reaction was performed as described inExample 1 using the 3′ blocked primer configurations of V1, V2-rU,V2-rC, V2-MM-rC, and V3-rC. Standard (non-blocked) primers were used asa control.

The RPA reaction was performed following the protocol in Example 1 using1 pM of template DNA (100 bp insert library with a tailed-A (57 bp) andP1B (53 bp) adapters with an expected amplicon size of about 210 bp),400 nm each of primers, 45 U or 20 U RNase HII enzyme (with 3′ blockedprimers) and 8 mM of Mg-acetate. After the reaction was performed andstopped, 15 uL of the 50 uL purified RPA reaction product was loadedinto wells of the agarose gel and the DNA fragments were separated byelectrophoresis.

Table 6 provides the identity of those samples loaded onto the gel. Asseen in FIG. 9 and summarized in Table 6, amplification products of theexpected size as well as primer dimers, were seen in amplificationsusing standard primers (lane 1). For the 3′ blocked primers, resultswere similar exemplary alternative reaction mixture to those obtainedusing the standard commercial reaction mixture. No reaction productswere detectable in samples generated using V1 or V3 primers, which havethe relatively short 3′ domain, or samples with 20 U of RNase HII or ano RNase control. All other samples that were generated using 3′ blockedprimers contained the expected amplification product in detectablequantities.

TABLE 6 Pellet formulation is compatible and results in efficient DNAtemplate amplification with the forward and reverse 3′ blocked primers.Lane Primer & Exp. Configuration Results M NEB Low MW ladder N/A 1Regular primers; TwistDx pellet Detectable amplification control productand nonspecific amplification product 2 3′ blocked primers, V2-rU;TwistDx Detectable amplification pellet control; 45U RNase H product 3Regular primers; alternative Detectable amplification exemplary RPAreaction mixture product formulation; 45U RNase H 4 3′ blocked primers,Vi; alternative No detectable exemplary RPA reaction mixtureamplification product formulation; 45U RNase H 5 3′ blocked primers,V2-rC; alternative Detectable exemplary RPA reaction mixtureamplification product formulation; 45U RNase H 6 3′ blocked primers,V2-MM-rC; Detectable alternative exemplary RPA reaction amplificationproduct mixture formulation; 45U RNase H 7 3′ blocked primers, V3-rC;alternative No detectable exemplary RPA reaction mixture amplificationproduct formulation; 45U RNase H 8 3′ blocked primers, V2-rU;alternative Detectable exemplary RPA reaction mixture amplificationproduct formulation; 45U RNase H 9 3′ blocked primers, V2-rU;alternative No detectable exemplary RPA reaction mixture amplificationproduct formulation; 20U RNaseH2 10 3′ blocked primers+, V2-rU; Nodetectable alternative exemplary RPA reaction amplification productmixture formulation; No RNase H (control)

In summary, the results confirmed that V1 and V3 primer configurations,which have a relatively short 3′ domain (4-6 nucleotides) length areinefficient in amplifying template DNA, but that V2 primers with eitherthe commercially available pellet and reaction mixture formulation orthe alternative exemplary RPA pellet and reaction mixture formulationefficiently amplified the template DNA. Furthermore, the experimentillustrates the robustness of the 3′ blocked primers to different basereaction mixture pellet and soluble reaction mixture formulations.Accordingly, In some embodiments, provided herein, is a reactionamplification method, such as RPA, performed using a dehydrated pelletformulation (which is rehydrated prior to use).

Example 5: Amplification of Template with RNase H Cleavable 3′ BlockedPrimers Attached to a Solid Support

This example illustrates clonal amplification of a DNA template using arecombinase polymerase amplification reaction wherein at least one ofthe primers of a primer pair is a 3′ blocked primer, wherein the 3′blocked primer is attached to a support. See FIG. 11 for exemplifiedprimers of the invention, wherein the blocking group C3 spacer isrepresented as 3spC3 in the primer sequences. The 3spC3 spacer caninclude phosphoramidite (available from Integrated DNA Technologies,Coralville, Iowa).

As a non-limiting example, the RPA method can be performed essentiallyusing the methods described in Example 1 in a single reaction vessel ina single continuous liquid phase, wherein a reverse 3′ blocked primerwith a V2 or V5 configuration is attached to a bead and the forwardprimer (3′ blocked primer of the invention or standard non-blockedprimer) is added in solution to the reaction mixture. Total reactionvolume is 50 μL to 1.2 mL. The following discusses a reaction in 300 μLvolume.

Beads, such as 1.25 μL from an 80 million/uL stock (100 million beads)with the attached reverse primer are added in a 1.5 mL tube (tube 1).The required bead count can vary depending on the sequencing chip(provided the bead-primer-template-MgOAc mixture volume does not exceed40% of the reaction volume) but is typically in the range from 20million to 1 billion beads for a reaction scale of 50 μL, up to 2.4 mL.For example, the bead count used can be from 10 to 100 million/μL. Aforward primer, standard or 3′ blocked primer of the invention, (1.2 μL,of a 100 μM stock) is added to the bead tube (tube 1), followed byvortexing and spinning to a final concentration of 0.4 μM in the finalreaction. The immobilized blocked reverse primer sequence can be one ofthe following:

(SEQ ID NO: 6) 5′-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAGCCrU CTC TAT GGG CAG TCG A/3 SpC3/-3′; (SEQ ID NO: 7)5′-CCA CTA CGC CTC CGC TTrU CCT CTC TAT GGG CAG /3SpC3/; or(SEQ ID NO: 8) 5′-C CTC CGC TTT CCT CTC TrAT GGG CAG TCG GTG AT /3SpC3/.

The forward primer of a standard (non-blocked) configuration, or a 3′blocked primer with a V5 configuration, is added to the bead tube (tube1), followed by vortexing and spinning. The solution forward primersequence can be one of the following:

(SEQ ID NO: 9) 5′-CCA TCT CAT CCC TGC GTG TC-3′; (SEQ ID NO: 10)5′-CCA TCT CAT CCC TGC GTG TCT CCG AC-3′; (SEQ ID NO: 11)5′-CCA TCT CAT CCC TGC rGTG TCT CCG ACT CAG /3SpC3/; or (SEQ ID NO: 12)AAC GAT CCA TCT CAT CCC TGC rGTG TCT CCG ACT CAG /3SpC3/

A biotinylated forward primer (0.12 uL of a 10 μM stock) is added to thebead tube (tube 1), followed by vortexing and spinning. The biotinylatedforward primer sequence can be: 5′Bio-CCA TCT CAT CCC TGC GTG TC-3′ (SEQID NO:13).

Various volumes of polynucleotide library (at 100 pM concentration) isadded to the bead tube (tube 1), followed by vortexing and spinning. Thelibrary volume can be varied depending on the desired DNA-to-bead ratioof 3:1, 2:1, 1:1, 1:1.7, 1:3, 1:5, 1:10. For example, the DNA-to-beadratio can be 1:1 to 1:1.5.

The rehydrated recombinase mix (tube 2, reconstituted in 180 μLrehydration buffer to a volume of approximately 185 μL) is added to thebead tube (tube 1), followed by vortexing and spinning.

Various amount of RNase H (at 20× concentrate from original NEB product)is added to the reaction tube, followed by vortexing and spinning. Thevolume is filled to 225 μL with low-TE.

75 μL of iced 28 mM Mg-acetate in sieving agent is added to the beadtube, followed by vortexing and spinning, and put back on ice for 10seconds, and incubated at 37° C. for 30-60 minutes on the heat block.

The reaction is stopped by adding 50 μL 500 mM EDTA, and 100 μL 1% SDS.The reaction tube is then topped off to 1 mL with TE buffer, followed byvortexing and spinning.

The beads are enriched by binding the biotinylated polynucleotides withparamagnetic beads conjugated with streptavidin (MyOne™ Bead fromDynabeads).

The enriched beads are loaded into an ION TORRENT ion-sensitive chip anda standard sequencing reaction is conducted.

The experiment disclosed above in this Example was performed using SEQID NO:6 attached to beads as the universal reverse 3′ blocked primer,SEQ ID NO:9 as the universal non-blocked standard primer in solution andSEQ ID NO:13 as the biotinylated forward primer for bead enrichment.Barcoded template DNA libraries (100 bp insert library with a tailed-A(57 bp) and P1B (53 bp) adapters with an expected amplicon size of about210 bp) (See FIG. 10) were used. Each of the barcoded libraries wasamplified in individual reactions, for a total of four samples, using 2to 4 uL of 20× RNase H enzyme, See FIG. 12A.

Following amplification, the beads from each barcoded library werecombined and sequenced on the same ION TORRENT sequencing chip. Theamplification of template was successful on beads immobilized with a 3′blocked primer from a reaction in a single continuous liquid phase. TheRPA amplification methods using an immobilized 3′ blocked primer of theinvention and RNase H enzyme generated monoclonally amplified templateson beads from a template library, which were subsequently sequenced. TheRNase H enzyme, which may be rate limiting for the amplification step,was analyzed by using two different volumes (2 μL and 4 μL) of the 20×enzyme (See FIG. 12A) for the impact downstream on the sequencingreaction. The estimated unit concentration of the 20× concentrate isabout 100 U/μl; 2 μL of RNase H contains about 200 U of enzyme and 4 μLcontains about 400 U of enzyme. The results indicated that more enzyme,as compared to half as much, resulted in better amplification of thetemplate on the beads. See FIG. 12B, wherein the samples (2 and 4) withabout 400 U of enzyme resulted in more “reads”, i.e. more amplifiedtemplate, as compared to samples 1 and 3 with half as much (200 U) RNaseH enzyme. Moreover, virtually complete amplification was reached within40 minutes. Little further gain was achieved with a longer 60 minuteamplification reaction. See FIGS. 12A to 12C.

Example 6: Amplification of Template with an Abasic (Baseless) CleavableBlocked Primer in a RPA Amplification Reaction

This example illustrates clonal amplification of a DNA template using arecombinase polymerase amplification reaction wherein at least one ofthe primers of a primer pair is a 3′ blocked primer, wherein the blockedprimer contains an abasic cleavable moiety that is apurinic,apyrimidinic or a spacer. That cleavable moiety is hydrolyzed by anapurinic/apyrimidinic (AP) endonuclease when the blocked primer ishybridized to the DNA template forming a double stranded duplex.Accordingly, the blocked primers comprise a 5′ domain, an abasiccleavable moiety (e.g. baseless), a 3′ domain and a blocking moiety(i.e. the primer is not extendable by a polymerase).

Class I and II AP endonucleases create a nick in the phosphodiesterbackbone at the 5′ side of the abasic site leaving a polymeraseextendable 3′-OH on the remaining 5′ domain of the primer for extension.Examples of AP endonucleases include endonuclease IV (commerciallyavailable from Thermo Fisher, Carlsbad, Calif.), APE 1 (commerciallyavailable from Thermo Fisher) and APE 2.

The blocked primers with a cleavable abasic site can be synthesized withan internal abasic furan (e.g., tetrahydrofuran) or spacer that replacesa nucleotide at the desired cleavage site. Alternatively, the primerscontaining an abasic site can be synthesized with a uracil basereplacing a nucleotide at the desired cleavage site. Prior to use in themethods, that primer can be treated with Uracil-DNA Glycosylase (UDG) toconvert the Uracil to an abasic site; UDG removes uracil residues fromthe sugar moiety of single- and double-stranded. DNA without destroyingthe phosphodiester backbone.

As a non-limiting example, the RPA method can be performed essentiallyusing the methods described in Example 1 in a single reaction vessel ina single continuous liquid phase, wherein a blocked (non-extendable)primer containing an abasic cleavable moiety and a biotin blockingmoiety with a V2 configuration is added in solution to the reactionmixture, wherein Endo IV or APE 1 are used as endonucleases replacingRNase H in Example 1. Amplification using the abasic cleavable blocked(non-extendable) primers with Endo IV or APE 1 was compared toamplification using ribobase cleavable blocked (non-extendable) primersof the invention with a V2 or V3 configuration and RNase H enzyme.

The abasic containing primers were synthesized with a uracil between the5′ domain and the 3′ domain of the blocked primers. The blocked forwardand reverse primer sequence were as follows, wherein the uracil isconverted to an abasic site prior to use:

Forward AP-15D: (SEQ ID NO: 28) GAA TCT GTC CAT AAG GTC AGT AAC GAT CCA U CT CAT CCC TGC GTG T-3′biotin Reverse AP-15D: (SEQ ID NO. 29)CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG CC U  CTCTAT GGG CAG TCG-3′biotin

The ribobase containing blocked primes used in a control RPA reactionwith RNase HII enzyme were selected from the primer pairs of SEQ ID NO.18 and SEQ ID NO. 19; SEQ ID NO. 20 and SEQ ID NO. 21; and SEQ ID NO. 23and SEQ ID NO. 24. See FIG. 10.

Prior to use in the RPA methods, the internal uracil of SEQ ID NO. 28and 29 was converted to an abasic site by treatment with UDG. 0.2 uL ofeach uracil-containing blocked primer (100 uM stock), 0.4 uL UDG (1U/uL, Thermo Fisher Scientific), and 1.2 uL RPA rehydration buffer (sameformulation as described for RPA reaction), were combined for a total of2 uL mixture. The mixture was incubated at 37° C. in a thermocycler for15 min for conversion to AP-containing primers. The 2 uL treated primermixture was then added to RPA reaction (50 uL reaction) as the primermix, so that each primer was 400 nM in the RPA reaction. The primer mixvolume can be adjusted for other desired primer concentrations.

Hence, the RPA method was performed using SEQ ID NO. 28 and 29,containing an abasic site at the internal uracil, and the APendonuclease endo IV (sourced from NEB and Thermo Fisher) or APE 1(NEB). Template DNA libraries with an expected amplicon size of about123 bp were used starting with 1 pM of template. Each of the librarieswas amplified in individual reactions, for a total of four samples,using 4 μL of commercial concentrations of Endo IV from NEB and ThermoFisher and 2 μL, or 5 μL of 10× Endo IV (Thermo Fisher). See FIG. 13.The RPA reaction mixture was incubated for 17 hours at 37° C.

The amplification of template was successful with a blocked primercontaining an abasic site from a reaction in a single continuous liquidphase when using 10× Endo IV. However, nonspecific products were alsopresent, along with the template. The RPA amplification method using aprimer with an abasic residue and endonuclease IV cleavage, could beoptimized to reduce or eliminate the nonspecific products. Amplificationof template was not successful when using Endo IV at the concentrationprovided by the commercial vendor. Not to be limited by theory, theresults indicate the Endo IV enzyme, similar to RNase H, may be ratelimiting for the amplification step, wherein more enzyme, i.e., 10×,resulted in better amplification of the template. In some embodiments,Endo IV is a viable endonuclease when paired with abasic 3′ blockedprimers for use in the RPA methods for amplification of template DNA.

The RPA method was also performed using SEQ ID NO. 28 and 29, containingan abasic site at the internal uracil, and the AP endonuclease APE 1(NEB) as compared to use of ribobase blocked primers (SEQ ID NO. 18 and19) and RNase H enzyme (NEB). Template DNA libraries with an expectedamplicon size of about 123 bp were used starting with 1 pM of template.Each of the libraries was amplified in individual reactions, for a totalof 10 samples, using 40 U of APE 1, 20 U or 45 U of RNase 100 nm to 800nm each of SEQ ID NO 18 and 19 for the RNase H containing reactionmixture and 100 nm or 400 nm of SEQ ID NO. 28 and 29 for the APE 1containing reaction mixture. See FIG. 14. The RPA reaction mixture wasincubated for 30 minutes at 37° C.

Amplification of the template was successful for all ten amplificationreactions including the reactions with the abasic cleavable blockedprimers and APE 1 enzyme. No non-specific amplification product wasobserved, including primer dimers. See FIG. 14. In some embodiments, APE1 is a viable endonuclease when paired with abasic blocked primers ofthe invention for use in the RPA methods for amplification of templateDNA.

A third experiment was conducted using RNase HII enzyme as a controlwith the primer pairs SEQ ID NO. 18 and 19; SEQ ID NO. 20 and 21 and SEQID NO. 22 and 23 (See FIG. 10) as compared with the use of Endo IV orAPE 1 enzyme and primer pair SEQ ID NO. 28 and 29, with and without theinternal uracil converted to a cleavable abasic moiety. The product fromthe above RPA reaction was obtained using 1 pM of template, as above,100 nm each of blocked primers, 20 U of RNase HII, 200 U or 50 U of EndoIV and 40 U of APE 1, wherein a portion of the 500_, purified RPAreaction product was loaded into wells of the agarose gels and the DNAfragments separated by electrophoresis. See FIG. 15. The RPA reactionmixture was incubated for 2 hours at 37° C.

The results indicate both Endo IV (when used at a 200 U concentration)and APE 1 are viable endonucleases, when paired with abasic blockedprimers of the invention, for the amplification of DNA template in a RPAamplification reaction. Non-specific product was observed with the useof Endo IV, see FIG. 15 at lane 6 of gel, but exonuclease activities ofthe enzyme or contamination of the commercial enzyme may be the sourceof the non-specific amplification. Accordingly, In some embodiments,provided herein, is a reaction amplification method, such as RPA,performed using abasic cleavable blocked primers and the endonucleaseAPE 1 or Endo IV for cleavage at the abasic site of the blocked primer.Following cleavage of the primer at the abasic site by either Endo IV orAPE 1, the 3′ end of the 5′ domain is extended and amplification of thetemplate results as disclosed herein for an RPA amplification reaction.

What is claimed is:
 1. A method for amplifying a nucleic acid template,comprising: a) forming a reaction mixture by combining a nucleic acidtemplate having a forward primer binding sequence and a reverse primerbinding sequence, a polymerase, a recombinase, a single-stranded bindingprotein, a recombinase loading protein, a forward primer that is notextendable by the polymerase, a reverse primer that is not extendable bythe polymerase, and an RNase H enzyme comprising a divalent cation,wherein the forward primer binding sequence is complementary oridentical to at least a portion of the forward primer and the reverseprimer binding sequence is complementary or identical to at least aportion of the reverse primer, and wherein the forward primer and thereverse primer comprise a 5′ domain and a 3′ domain separated by anucleotide comprising one or more nucleotides that are cleavable by theRNase H enzyme, wherein the 5′ domain is 10 to 40 nucleotides in lengthand the 3′ domain is 10 to 25 nucleotides in length; and b) incubatingthe reaction mixture under substantially isothermal amplificationconditions between 35° C. and 45° C. for 15 minutes to 60 minutes,thereby amplifying the nucleic acid template.
 2. The method of claim 29,wherein the nucleic acid template is a member of a nucleic acid librarycomprising a population of nucleic acid templates each comprising aforward primer binding sequence, and wherein the forward primer is auniversal forward primer that binds the universal forward primer bindingsequence.
 3. The method of claim 30, wherein the nucleic acid templateseach comprise a reverse universal primer binding sequence and whereinthe reverse primer is a universal reverse primer that binds theuniversal reverse primer binding sequence.
 4. The method of claim 29,wherein either or both of the forward primer and the reverse primer areimmobilized on a solid support.
 5. The method of claim 32, wherein thesolid support is a bead.
 6. The method of claim 29, wherein the 5′domain is 15 to 30 nucleotides in length.
 7. The method of claim 29,wherein the 3′ domain is 14 to 25 nucleotides in length.
 8. The methodof claim 29, wherein the 3′ domain is 15 to 25 nucleotides in length. 9.The method of claim 36, wherein a 3′ nucleotide of the 3′ domain of theforward primer is mismatched to the forward primer binding sequence. 10.The method of claim 29, wherein the RNase H enzyme is E. coli RNase HII.11. The method of claim 29, wherein the reaction mixture comprises uvsY.12. The method of claim 29, wherein the recombinase is selected from thegroup consisting of uvsX, RecA, RadA, RadB, Rad 51, a homologue thereof,a functional analog thereof and a combination thereof.
 13. The method ofclaim 29, wherein the reaction mixture comprises uvsY accessory proteinand uvsX recombinase.
 14. The method of claim 29, wherein the amplifyingis from 15 to 45 minutes.
 15. The method of claim 29, wherein the RNaseH enzyme is RNase HII and the incubating temperature is between 35° C.and 40° C.
 16. The method of claim 29, wherein RNase H enzyme is presentat a concentration from 5 U to 200 U/50 μL.
 17. The method of claim 29,wherein the RNase H enzyme is present at a concentration from 10 to 90U/50 μL.
 18. The method of claim 29, wherein the reaction mixturecomprises magnesium or manganese ions.
 19. The method of claim 29,wherein the one or more nucleotides that are cleavable by the RNase HEnzyme is rU, rG or rA.
 20. The method of claim 29, wherein the 3′domain of the non-extendable primers is 14 to 20 nucleotides in lengthand the one or more nucleotides that are cleavable by the RNase H enzymeis rU, rG or rA.
 21. The method of claim 29, wherein the polymerase isSau polymerase, T7 polymerase with reduced 3′ to 5′ exonucleaseactivity, Bsu polymerase, or a combination thereof.